CN1346887A - Production and transport of protein - Google Patents

Abstract

The invention relates to constructs comprising: a) a targeting sequence; b) a regulatory sequence; c) an exon; and d) an unpaired splice-donor site. The invention further relates to a method of producing protein in vitro or in vivo comprising the homologous recombination of a construct as described above within a cell. The homologously recombinant cell is then maintained under conditions which will permit transcription and translation, resulting in protein expression. The present invention further relates to homologously recombinant cells, including primary, secondary, or immortalized vertebrate cells, methods of making the cells, methods of homologous recombination to produce fusion genes, methods of altering gene expression in the cells, and methods of making a protein in a cell employing the constructs of the invention.

Description

Proteinic production and proteinic conveying

In recent years developed the method for using therapeutic protein treatment disease, it comprises the treatment protein of produced in vitro for conventional medicine conveying (for example intravenous injection, subcutaneous injection or intramuscularly), and has set up gene therapy method recently.

The protein of tool therapeutic activity generally imports in the suitable cell by the proteinic foreign DNA of therapeutic activity of will encoding and prepares.For example, with the proteinic foreign DNA of the required treatment of coding, import in the carrier of plasmid and so in the cell as the permanence cell, the protein that is encoded is therein expressed.And existing people proposes, and foreign cell gene and expression thereof can be hit target by gene and be modified, for example referring to U.S. Patent No. 527071, WO91/06666, WO91/06667 and WO90/11354.

Present admissible gene therapy method is to utilize infectious vector, retrovirus vector for example, and it comprises the genetic material that desire is expressed.This method has limitation, for example may produce the virus of tool replication at the carrier production period: the reorganization between therapeutic virus and the endogenous retrovirus genome might produce and has new cell-specific, host range or increased toxicity and Cytotoxic infectant; Independently be incorporated in a large amount of cells, increased tumorigenesis and inserted active danger; The limited clone's ability (it has limited the treatment suitability) and the short life expression in vivo of useful products in the retrovirus.A kind of method that gene product is provided preferably, particularly a kind of limitation and the dangerous method that can avoid present method therefor to be associated is valuable.

The present invention relates to treatment and produce and transmit treatment protein improvement method with proteinic produced in vitro and by gene therapy.In the method, the expression of required target gene in cell (being a kind of required endogenous cell gene), by will comprising the DNA that regulates sequence, exon and splice donor site at least, giving selected earlier site in homologous recombination transfered cell genome and change.These compositions import among karyomit(e) (group group) DNA in such a way, promptly will make it to cause producing new transcription unit (the adjusting sequence, exon and the splice donor site that wherein are present in the DNA construct are operably connected on the native gene).Because these compositions are introduced in the chromosomal DNA, have consequently changed the expression of required native gene.

As used herein, the genetic expression that changes comprises that activation (perhaps making it to express) is in the gene of static (not expressing) state usually when obtaining in cell, improving can not be with the expression of gene level of significance level expression on the physiology in cell when obtaining, change and regulate or inducing properties, so that it occurs in cell when obtaining is different, and the gene expression dose of in cell, expressing when reducing (comprising eliminations) former acquisition.

The invention still further relates to the DNA construct that is applicable in the method that changes expression of target gene.This DNA construct comprises: (a) hit target sequence; (b) regulate sequence; (c) exon; (d) unpaired splice donor site.This hits target sequence tutorial element (a)～(d) and is integrated in the intracellular target gene in the DNA construct so that element (b) but～(d) be connected on the endogenous target gene sequence with operating method.In another embodiment, this DNA construct comprises: (a) hit target sequence, (b) regulate sequence, (c) exon, (d) splice donor site, (e) intron, and (f) acceptor splicing site site, wherein hit the integration of target sequence tutorial element (a)～(f) so that element (b) but～(f) be connected on the native gene with operating method.Hit target sequence and be with cell chromosome DNA (homologous recombination takes place by it) in give and select the site homologous.In this construct, exon generally be regulate 3 of sequence ', and splice donor site be 3 of interior apparent son '.

Following content is used to describe two embodiments of the present invention, wherein the sequence upstream with human erythropoietin (hEPO) gene changes, so that hEPO can be expressed in elementary, secondary or permanence cell, and these cells are in firm acquisition, can not reach EPO with detectable scale when being in not transfected state.In first embodiment, hit the target construct and contain two and hit target sequence.First hits target sequence and comes from the second 5 ' sequence of hitting target sequence together, and two sequences are upstreams of hEPO coding region.Hit the target construct and also contain regulation domain (mMT-1 promotor), exon (human growth hormone (hGH) exons 1) and unpaired splice donor site.Having this homologous recombination product that hits the target construct is shown among Fig. 1.

In the embodiment 2, hit the target construct and also contain two and hit target sequence.First hits the sequence homology in target sequence and the endogenous hEPO regulation domain, and second hit and show sub 1 homology in target sequence and the hEPO.Hit the target construct and also comprise regulation domain (mMT-1 promotor), exon (hGH exons 1) and unpaired splice donor site.Have this homologous recombination product that hits the target construct and be shown in Fig. 2.

In two embodiments, the product that hits the target process is a chimeric transcription unit, first exon that this chimeric transcription unit produces a kind of hGH gene is positioned the ripe mRNA of hEPO exon 2～5 upstreams, the product of transcribing, splicing and translate is a kind of protein, and wherein the amino acid/11 of hEPO signal peptide～4 are replaced by the amino-acid residue 1～3 of hGH.These two embodiments, at the relative position of the relevant adjusting sequence of hitting the target construct that is inserted into, the splicing specificity mode aspect of the processed transcript required appearance last with being generation is different.

The invention still further relates to by homologous recombination, above-mentioned construct is imported among the host cell chromosome DNA and produce the homologous recombination somatocyte in external or body, to produce method of protein.Then this homologous recombination somatocyte is maintained can make its transcribe, translate with the excretory condition under, thereby produce useful proteins matter.

The present invention relates to be used to produce protein, the transfected cell of therapeutic protein particularly, for example transfected elementary or secondary cell (being impermanentization cell) and transfected permanence cell, make this class cell method, use this class cell in produced in vitro method of protein and gene therapy methods.Cell of the present invention derives from vertebrates, Mammals particularly, more specifically be to derive from the people.The cell that produces by method of the present invention contain coding treatment product DNA, itself be the DNA of treatment product and/or can cause transfected cell high level expression gene or have and be different from the corresponding not adjusting that transfected cell occurred or induce the DNA of mode.

The invention still further relates to so as to making cell, for example elementary, secondary and permanence cell transfected with the method that comprises exogenous genetic material, produce the method for clonal cell line or allos cell strain and use transfected elementary, secondary or permanence cellular immunization animal, or in the immunized animal body, produce the method for antibody.

The present invention be more particularly directed in eukaryotic cell, for example fungi, plant or animal vertebrates for example particularly feeds animal, and more particularly gene hits the method for target or homologous recombination in the cell in people source.Promptly relate to by homologous recombination, DNA is imported the method in elementary, the secondary or permanence cell in vertebrates source, this DNA is imported into giving of elementary, secondary or permanence cell genomic dna and selects the site like this.The used target sequence that hits can be selected with reference to hitting the site that DNA prepares to insert in the target DNA construct.The invention still further relates to elementary, the secondary or permanence cell of producing by method of the present invention of homologous recombination body, be referred to as elementary, the secondary or permanence cell of homologous recombination body (HR), and HR is elementary, secondary or the use of permanence cell.

In one embodiment of the invention, expression of gene is changed, and gene is activated.Promptly be present in the primary and secondary of vertebrates source or the permanence cell, the gene of not expressed in archeocyte usually during acquisition is activated, and coded protein is expressed as a result.In this embodiment, use homologous recombination with replace, impairment or destroy regulation domain, this zone usually with can cause that by insertion the cytogene that the adjusting sequence of gene with the horizontal expression that is higher than corresponding not transfected cell obtains is relevant.

In one embodiment, the gene that is activated can further be increased by the method that comprises the alternative marker gene of an amplification property, this marker gene is that such character is arranged, but promptly contain the cell of the copy that increases of selected marker gene, can be selected by culturing cell in the presence of the suitable factor selected.But the activated native gene is to be amplified in placed in-line mode with amplification property selected marker gene.The cell that contains many copies of the native gene that is activated can be used for produced in vitro protein and gene therapy.

As of the present invention, gene hits target and amplification effect is useful especially to activating the expression of gene that forms transcription unit, said transcription unit is enough greatly to cause it to be difficult to separate and to express, perhaps for activating that whole protein coding zone can not utilize or being not useful especially by cloned genes as yet.

In another embodiment, the expression when the expression of gene ratio has just obtained in cell is improved, and perhaps demonstrates to be different from adjusting or the induced character that occurs under the corresponding not transfected cell situation.Expression level low (reducing or eliminating) when in yet another embodiment, expression of gene is than acquisition just in cell.Be used for external protein production in yeast or the bacterium for transferring to, the present invention has also described and has utilized homologous recombination that genetic modification is become not have the interior method that shows the cDNA copy of son.

Transfected cell of the present invention has a lot of purposes in humans and animals.In one embodiment, in this cell human implantable or the animal body, so as in human body or animal body, carrying protein.For example, be therapeutic purpose, can be with hGH, hEPO, people's pancreotropic hormone and other protein whole body or be transported in the human body partly.In addition, also can produce generation tethelin, plain, the proteinic transfected inhuman cell of pancreotropic hormone and other inhuman source of short cell generation.

The barrier device (but by this device treatment product free permeation) that contains the transfected cell of expression treatment product can be used in body internal fixing position limit cell, or protects protection and isolated cell from host immune system.Barrier device is of great use, and it can make transfected permanence cell, transfected heterogenous cell or transfected homogeneous variant cell implanted, with treatment human or animal's illness or be applied to agricultural (for example produce long hormone and be used for milk-producing).When making methods of treatment be subjected to stoping because of some reason, barrier device can carry out short-term (being temporary transient) treatment easily because the approaching easily cell that will remove can be provided.In addition, transfected xenogenesis and homogeneous variant cell can not have to be used to the short-term gene therapy under the situation of barrier device, and the gene product that is produced by cell will be transferred in vivo like this, till cell is repelled by host's immune system.

Transfected cell of the present invention also can be used for exciting antibody to produce, or immune humans and animals is with the antagonism virulence factor.The transfected cell of implanting can be used for carrying the immunizing antigen of energy stimulation of host cell and humoral immune reaction.These immune responses can design protects the host to infect (being immune fitting) to avoid following infectant; exciting and to improve resistance against diseases, or produce the antigenic antibody that produces in vivo at by the transfected cell that can be used for treating with diagnostic purpose at the infection of continuous development.The removable barrier device that contains this cell can be used as termination and stands antigenic simple tool.In addition, the cell of being ostracised the most at last (the transfected cell of xenogenesis or allogeneic) also can be used for restriction and stands antigen, because when cell is ostracised, antigenic generation will stop.

Method of the present invention can be used to produce elementary, the secondary or permanence cell that can produce the useful product of various widely treatment and comprises (but not being only limited to): hormone, cytokinin, antigen, antibody, enzyme, thrombin, transport protein matter, acceptor, adjusting protein, structural protein, transcription factor, ribozyme (ribozymes) or sense-rna.In addition, method of the present invention also can adopt the cell of ribozyme (ribozy-mes), protein or the nucleic acid (they are used to external generation treatment product or are used for gene therapy) of the appearance of production capacity generation in next life non-natural.

Brief Description Of Drawings

Fig. 1 is the synoptic diagram of expression transcriptional activation hEPO genetic method; Thick line: mouse metal sulfur hydrochloride I promotor; Vertical bar line square frame: 5 of hGH ' does not translate the district; Filled box: hGH exons 1; Horizontal stripe line square frame: the 10bP splicing donor sequences of hEPO introne 1; 5 of cross hatch square frame: hEPO ' does not translate the district; Hollow numbering square frame: hEPO encoding sequence; Draw oblique line square frame: hEPO 3 ' and do not translate sequence; The HIII:HindIII site.

Fig. 2 is the synoptic diagram that shows transcriptional activation hEPO genetic method; Thick line: mouse metal sulfur hydrochloride I promotor; Vertical bar line square frame: 5 of hGH ' does not translate the district; Filled box: hGH exon I; Hollow numbering square frame: hEPO encoding sequence; Draw slanted bar line square frame: hEPO 3 ' does not translate sequence; The HIII:HindIII site.

Fig. 3 represents the synoptic diagram of plasmid pXGH5, and this plasmid comprises the hGH gene that is under the control of mouse metal sulfur hydrochloride promotor.

Fig. 4 represents the synoptic diagram of plasmid pE3neoEPO.Wherein pointed out the position of human erythropoietin gene and neomycin phosphotransferase gene (neo) and penbritin (amp) resistant gene.Arrow is indicated each gene transcription direction.PmMT1 represents mouse metal sulfur hydrochloride promotor (driving hEPO expresses), and pTK represents herpes simplex virus thymidine kinase promotor (driving neo expresses).The gene map midpoint area is represented from person-time position of the on-site sequence of xanthine-guanine phosphoribosyl transferase (HPRT).Wherein marked the relative position of restriction endonuclease recognition site.

Fig. 5 represents the synoptic diagram of plasmid pcDNEO, this plasmid comprises the neo coding region from plasmid pSV2neo (BamHI-BglII fragment) in the BamHI site that is inserted into plasmid pcD, from Amp-R and the pBR 322 Ori sequences of pBR 322, and polyA, 16S splicing bonding land and from the early promoter zone of SV40.

Fig. 6 represents plasmid pREPO4 synoptic diagram.

The erythropoietin that Fig. 7 is shown in the human cell line that hits who stands progressively to select in 0.02,0.05,0.1, the 0.2 and 0.4 μ M methotrexate is expressed.

Fig. 8 represents the synoptic diagram of plasmid pREPO15.The filling frame table shows the fragment from genome hEPO sequence.Between BamHI (3537) and BglII '/HindIII ' zone is equivalent to Genba-nK and charges to the sequence on 1～4008 among the HUMERPALU.Zone between BglII '/HindIII ' (11463) is equivalent to Genbank and charges to the dna sequence dna on 4009～5169 among the HUMERPALU.Zone between HindIII (11463) and XhoI (624) is contained and is equivalent to 7～624 the sequence that Genbank charges to HUMEPA.Hollow frame is represented the CMV promoter sequence, wherein contains the sequence from Nucleotide 546～2105 among the Genbank sequence HS5MIEP.The hollow frame of band arrow is represented the neo gene.Draw the hachure frame table and show that the thymidine that drives neo genetic expression swashs nurse (tk) promotor.Fine rule represents to comprise the pBSIISK+ sequence of amp gene.

Fig. 9 A lists restriction map, and observed product (top) behind the diagram digestion endogenous hEPO gene, and with the activated hEPO gene (below) after the target fragment homologous recombination that hits of pREO15.

Fig. 9 B lists (T1) human fibroblasts of being untreated (HF) and hitting is cloned the result (referring to embodiment 7) who carries out digestion with restriction enzyme and Southern hybridization analysis in HF342～15.

Figure 10 represents the synoptic diagram of plasmid pREPO18.Fill frame and represent the fragment that derives from genome hEPO sequence.Zone between BamHI (3537) and ClaI (7554) is equivalent to Genbank and charges to the sequence on 1～4008 among the HUMERPALU.Zone between ATG (12246) and HindIII (13426) is equivalent to Genbank and charges to the dna sequence dna on 4009～5169 among the HUMERPLAU.Zone between HindIII (13426) and the XhoI (624) is contained and is equivalent to the sequence that Genbank charges among the HUMERPA 7～624.The CMV promoter sequence represents with hollow frame, and contains the sequence from Nucleotide 546～2015 among the Genbank sequence HS5MIEP.Tetrahydrofolate dehydrogenase (dhfr) transcription unit shows with the some frame table of band arrow.The neo gene is represented with the hollow frame of band arrow.The tk promotor that drives the neo gene is shown to draw the hachure frame table.The fine rule representative contains the pBSIISK+ sequence of amp gene.

Figure 11 illustrates the intronless gene that is used for activating and increasing of the present invention, the construct of alpha-IFN gene, wherein this construct comprises: first hit target sequence (1), the marker gene that can increase (AM) but selected marker gene (SM), regulate sequence, CAP site, splice donor site (SD), intron (fine rule), acceptor splicing site site (SA) and second and hit target sequence (2).Black surround is represented coding DNA, and sequence is not translated in the representative of some frame.

Figure 12 illustrates that the present invention is used to activate and the construct of the endogenous gene that increases, wherein first exon impels signal peptide, human GM-CSF gene, described construct comprise first hit target sequence (1), the marker gene that can increase (AM) but selected marker gene (SM), regulate sequence, CAP site, splice donor site (SD) and second and hit target sequence (2).Dark square is represented coding DNA and is put the square frame representative and do not translate sequence.

Figure 13 illustrates that the present invention is used to activate and the construct of the endogenous gene that increases, wherein first exon impels signal peptide, human G-CSF gene, this construct comprises and first hits target sequence (1), the marker gene (AM) that can increase, selectable marker gene (SM), regulates sequence, CAP site, and splice donor site (SD) and second hits target sequence (2).Dark square is represented coding DNA and is spent the representative of some square frame not translate sequence.

Figure 14 illustrates that the present invention is used to activate and the construct of the endogenous gene that increases, wherein first exon is non-coding, people FSH β gene, this construct comprises first and hits target sequence (1), the marker gene that can increase (AM) but selected marker gene (SM), regulate sequence, CAP site, splice donor site (SD) and second and hit target sequence (2).Black surround is represented coding DNA and is put the frame representative and do not translate sequence.

The present invention is based on discovery and can be binned in the cytogene group preliminary election site by homology and inserts The construct that enters DNA changes regulating action or its activity of useful endogenous gene in the cell, Described construct comprises: (a) hit target sequence; (b) regulate sequence; (c) extron and (d) non-The pairing splice donor site wherein hits the integration that target sequence instructs element (a)～(d), so that Element composition (b)～(d) be operably connected on the endogenous gene. In another embodiment side In the case, DNA construct comprises: (a) hit target sequence, (b) regulate sequence, and (c) extron, (d) splice donor site, (e) introne and (f) acceptor splicing site site wherein hit the target order Row instruct the integration of element (a)～(f), so that in element (b)～(f) is operably connected to On the First Exon of source property gene. Can according to the site that is inserted into DNA select use Hit target sequence. In two embodiments, hit the target process for generation of new transcript unit, It is a kind of fusion product of the sequence by hitting the importing of target DNA construct and endogenous cellular gene Thing. As discussed in this article, for example can be by in the genomic DNA of host cell, leading Enter construct of the present invention to form new transcript unit, transcribe silent gene (namely thereby make Can not be at the cell inner expression gene before the transfection) in host cell, be activated. Also such as this Literary composition is discussed, but the method for the application of the invention and DNA construct change at cell The expression of the endogenous gene of interior expression wherein can be raising, the reduction bag of expression Draw together elimination, perhaps can change and regulate or induced character.

As mentioned above, the present invention relates to come source cell with eucaryon, as fungi, plant or Animal, such as vertebrate, mammal particularly is more specifically in the cell in people source Gene or the DNA method of hitting target. In other words, the present invention relates to recombinate by the homology of DNA Or hit cell such as elementary, the secondary or permanence cell that target imports DNA in vertebrate source Interior method, wherein said DNA is in the genomic DNA of the pre-site transfered cell of selecting. More particularly, the present invention relates to homology restructuring, wherein by use comprise hit target sequence, The DNA construct of adjusting sequence, extron and splice donor site is modified endogenous gene Transcribe and/or translational product. It is thin to the invention still further relates to the homology recombinant that produces by this method The application of born of the same parents and homology recombinant cell.

The present invention also relates to activate the primary cell, the secondary cell that are present in the vertebrate source Or in the permanence cell, but usually not in the method for the gene of these cells. Use Homology is recombinated or is hit target with in the genome sequence with the DNA transfered cell, causes gene being subjected to Expressed in the body cell. In another embodiment, by importing DNA construct, carry Adjusting or the induced character of the expression of high gene gene in cell or change gene. Knot Fruit makes the product that is encoded to be expressed than level higher in corresponding non-transfected cell. This Inventive method and DNA construct also can be used for producing such cell, i.e. required product wherein Low at corresponding not transfected intracellular expression at transfected intracellular expression ratio. Just Be to say, in the resulting archaeocyte of transfected cell internal ratio, produce protein (bag still less Draw together nonprotein).

In another embodiment, encode the normal silent gene of required product transfected Be activated and increase in elementary, the secondary or permanence cell. This embodiment is a kind of passing through Import common on function, the connection with endogenous gene with the restructuring of genomic DNA homology The method of dna sequence dna, wherein (1) when this dna sequence dna the endogenous gene place or near quilt insert When entering in the host gene group, can be in order to changing the expression of (as activating) endogenous gene, and And the cell of (2) being selected those endogenous genes that wherein activate to be amplified. In addition, exist The expression of the gene of usually expressing in the gained archaeocyte is improved and gene is also expanded Increase.

Hereinafter describe DNA construct of the present invention, produce transfected thin with these DNA constructs The application of born of the same parents' method, transfectional cell and these cells.

DNA construct

DNA construct of the present invention comprises following composition at least: hit target sequence, regulate sequence, Extron and the splice donor site that do not match. In this construct, extron is being regulated sequence 3 ' side, and do not match the 3 ' side of splice donor site at extron. In addition, have at side joint Do not match the extron front (5 ' direction) of splice donor site can have a plurality of extrons and / or introne. As discussed herein, additional construct composition is arranged usually, as optional Select the sign sign that maybe can increase.

DNA can be called as ectogenic in the construct. Term used herein " exogenous " Be defined as by method of the present invention, import to such as the DNA construct that limits by this paper Intracellular DNA. Exogenous DNA can have and be present in intracellular endogenous before the transfection The sequence that DNA is identical or different.

Hit target sequence

Hit target sequence and be and allow the recombinate selected cell that contains useful gene of reasonable homology Dna sequence dna in the genome. Hit target sequence generally with normal presence in the gained archaeocyte Dna sequence dna in the genome (as is positioned at transcription initiation site upstream, the transfection of useful gene Coding or the noncoding DNA in the termination site or downstream are perhaps deposited by original modification Be the sequence in the genome) homology (namely identical with cell DNA or have enough similar, The homology restructuring can take place so that hit target sequence and cell DNA) dna sequence dna. Can be with reference to inciting somebody to action Select the used target sequence that hits to the site of the DNA that wherein inserts DNA construct.

Can utilize one or more target sequences that hit. For example, circular plasmids or dna fragmentation are preferred Use the single target sequence that hits. Linear plasmid or dna fragmentation preferably use two to hit target sequence. Hit target sequence can be at random useful gene (such as the sequence of extron and/or introne) it In, directly be contiguous on the useful gene (namely in the code area of hitting target sequence and useful gene it Between do not have other nucleotides), in the upstream of useful gene (such as the order of upstream noncoding region Row or endogenous promoter sequence) or in the upstream of gene and leave certain distance (as The upstream sequence of endogenous promoter). Hit target sequence can comprise those at present known or The quilt of having measured sequence hits the zone of gene, and/or does not determine on the structure but can make The more upstream of making gene map and being determined by these those skilled in the art with restriction enzyme The territory.

As teaching herein, available gene hit Target process will separate from heterogeneic, by Separation from the assembling of the composition of different cells and/or viral source or as new adjusting order Row with the synthetic adjusting sequence of gene engineering method be inserted in the endogenous cell gene, with it straight Connect adjacent, upstream or have with it in the site of suitable distance. In addition, can be by hitting target Replace, remove, add or modify the structure that can affect the RNA that produces or protein or steady Sequence qualitatively. For example, can modify the RNA molecule rna stability element, splicing site and / or targeting sequencing, to improve or to change function, the stability of RNA molecule and/or translate energy Power. Also can change protein sequence such as burst, former peptide sequence, avtive spot and/ Or structure sequence is to improve or to modify transportation, secretion or the functional characteristic of protein. According to this Method imports normal expression character and/or protein or RNA that foreign DNA can cause gene The change of architectural characteristic.

Regulate sequence

The adjusting sequence of DNA construct can by one or more promoters (as consist of or can The promoter of inducing), enhancer, skeleton bonding pad or matrix connect site, the negative unit that regulates The bond of part, transcription factor binding site point or described sequence.

Regulate sequence and can comprise derivable promoter, can be imported into individuality during so former generation Cell when interior not expression product can be induced to express (in transfected cell generation Rear but be induced expression before implanting or after implanting). Certainly, can advance when importing The mode that row is expressed (for example, under the promoter condition that consists of) the required product of will encoding In the DNA transfered cell. Can be from cell or viral genome the separation adjusting sequence, such Regulating sequence comprises and regulates the early stage or late gene of SV40, the big late gene of adenovirus, mouse Metallothioneins-I gene, elongation factor-1 α gene, the huge viral gene of cell, collagen egg White gene, actin gene, immunoglobulin gene or HGM-CoA reductase gene table The sequence that reaches. Regulate sequence and contain preferably the transcription factor binding site point, as the TATA box, CCAAT box, AP1, SP1 or NF-KB binding site.

Additional DNA construct element

DNA construct also comprises one or more extrons. Herein extron is defined as The dna sequence dna that is copied into RNA and exists with ripe mRNA molecule. Extron optionally contains It (is codon that coding one or more amino acid and/or certain seed amino acid of partly encoding are arranged One or two base) DNA. In addition, extron also contains and is equivalent to 5 ' noncoding region DNA. Such as the one or more amino acid of exogenous exons coding and/or certain amino acid whose Part, design dna construct so that make when transcribing and splice are understood the of code and target gene Two extrons or code area are in the code. Term used herein " in the code " refers to when the When the coded sequence of one extron and Second Exon merges, nucleotides just with do not change by The suitable mode of understanding code of the mRNA part that Second Exon is derived links together.

First Exon such as middle target gene contains the sequence ATG that translates in order to startup, then structure The exogenous extron of building body preferably contains ATG, and also contains one or many in case of necessity Individual nucleotides, thus make second of target gene in the comprising of gained and continue after extron The mRNA code area is in code. This wherein First Exon contains the target gene of ATG Example comprises granulocyte/macrophage colony stimulatory factor (hGM of coding hEPO, hGH, people-CSF) and the gene of people's granulocyte colony stimulating factor (hG-CSF).

As target gene coding erythropoietin, exogenous exon can be derived from any gene, and exon comprises the part of ATG initiator codon and coded protein in the described gene.Exon further terminates in first Nucleotide place of codon.Example comprises apolipoprotein E gene (exons 1 and 2), placenta prolactin antagonist gene (by hCS-A and hCS-B genes encoding), prolactin antagonist gene, human growth hormone variant gene (hGH-B), human placental lactogen-L gene (hCS-L) and granulocyte/macrophage colony stimulating factor gene (EM-CSF).

Splice donor site is to instruct the sequence of an exon splicing to another exon.Usually, first exon is positioned at 5 ' side of second exon, and overlapping and side joint first exon is discerned the acceptor splicing site site of side joint second exon on second exon, 5 ' side at the splice donor site of first exon on its 3 ' side.It is the characteristic of the representative sequence that conforms to that splice donor site has with (A/C) AGGURAGU (wherein R represents purine nucleotides), and requiring on the 4th and the 5th is GU (Jakson, I.J., Nucleic Acids Research 19:3715-3798 (1991).Conform to first three base in site of splicing donor is back three bases of exon.Splice donor site is to be limited by the ability that they influence appropriate reaction in the mRNA splicing approach on function.

The splice donor site that do not match herein is defined as being present in and hits in the target construct and with in the construct be not positioned at the splice donor site that accompanies in this acceptor splicing site site of not matching splice donor site 3 ' side.The splice donor site that do not match causes the splicing with endogenous acceptor splicing site site.

The same with splice donor site, the acceptor splicing site site in the sequence also is the splicing of instructing an exon and another exon.Based on the effect of performance during splice donor site combines, splicing body uses the acceptor splicing site site to remove intron.The acceptor splicing site site has the characteristic sequence with YYYYYYYYYYNYAG representative, and wherein Y represents any pyrimidine bases and N represents any Nucleotide (Jackon, I.J., Nacleic Acids Research 19:3715-3798 (1991)).

Intron is defined as between two exons, and the sequence of one or more Nucleotide of removing from precursor rna molecule by splicing in mRNA molecule forming process.

Regulating sequence is for example to be operably connected on the ATG initiator codon, and it can start translates.Optionally, CAP site (link to each other with regulatory region and be conditioned the specific mrna initiation site that the district utilizes) is operably connected to and regulates on sequence and the ATG initiator codon.In addition, and regulate that sequence links to each other and this CAP site of being conditioned the sequence utilization is not included in and hits in the target construct, and transcribe mechanism and will limit a new CAP site.For most of genes, the CAP site sees about 25 the Nucleotide places of TATA box 3 ' side usually.In one embodiment, splice donor site is positioned at and ATG direct neighbor place, for example when second exon of exogenous exon and target gene be that just not necessarily there are one or more Nucleotide in requirement when coexisting in the sign indicating number.Encode the encoding sequence of the DNA of one or more amino acid or amino acid moiety and target gene in sign indicating number the time, and then it preferably is positioned at directly and the ATG adjacent on its 3 ' side.In such embodiment, splice donor site is positioned at directly adjacent with the coding DNA on its 3 ' side.

That settles on that be operably connected or the function is meant a kind of configuration, wherein exogenous adjusting sequence, exon, splice donor site, randomly certain sequence of Cun Zaiing and acceptor splicing site site suitably hit target with the corresponding position of endogenous gene on, thereby make regulatory element instruct the generation of elementary rna transcription thing, this transcript to begin (can randomly be included in and hit in the target construct) in the CAP site and comprise the sequence of the exon and the splice donor site that are equivalent to hit the target construct, be positioned at endogenous gene and regulate the DNA of regulatory region (if present) upstream, the regulatory region of endogenous gene (if present), endogenous its because of 5 ' nontranscribed domain (if present), exon and intron (if present) with endogenous gene.In the configuration that is operably connected, the splice donor site that hits the target construct instructs a process of splicing mutually with the acceptor splicing site site of one of exon of side joint endogenous gene, thus the required protein of mature transcript generation that can connect from spelling.In one embodiment, the acceptor splicing site site is endogenic, and splicing is at the endogenous exon like this, for example the endogenous exon of native gene.In another embodiment, the acceptor splicing site site is included in to be hit in the target construct, and this splicing can be removed by hitting the intron that the target construct imports.

Used coding DNA (as in the exons 1 that hits the target construct) one or more amino acid of can randomly encoding, and/or certain amino acid whose part, they are same with the coding DNA of endogenous protein.DNA sequences encoding used herein for example can be corresponding to first exon of useful gene.This coding DNA codified is different from one or more amino acid or certain amino acid whose part of useful proteins first exon in addition.Proteinic one or more activity are not that such embodiment is significant especially under the very crucial situation for this when the amino acid of useful proteinic first exon.For example, when making up, then can utilize the sequence of first exon of coding hGH with endogenous hEPO gene formation syzygy.In this example, the fusion of hGH exons 1 and hEPO exon 2 causes having the formation of the active heterozygosis signal peptide of function.In relevant construct, can use the amino acid that wherein is encoded not hinder anyone or the exon in inhuman source of the function of heterozygosis signal peptide.In a related embodiment, also can use this technology to see sudden change in the target gene with correction.

As required product is endogenous protein and when hitting the fused protein of encoding sequence in the target construct, be incorporated into intracellular exogenous coding DNA by present method and comprise the DNA of one or more exons that encodes, or be equivalent to be fused to translating or the cDNA sequence of transcription product on the endogenous target gene product.In this embodiment, use is hit Target process and is prepared chimeric or multifunctional protein, and it will be combined in the polypeptide from two or more proteinic structures, enzymatic or aglucon or receptors bind character.For example, the foreign DNA codified is localized anchorage peptide or signal peptide on the target protein plasma membrane, to provide or to improve emiocytosis, leader sequence, enzymatic zone, stride film dominant area, cofactor calmodulin binding domain CaM or other functional regions.Do not secrete under the normal circumstances but can merge to provide excretory protein example to comprise dopa decarboxylase, transcription regulatory protein, alpha-galactosidase and white horse with a black mane propylhomoserin hydroxylase with signal-proteins.

First exon as target gene is equivalent to non-coding region (for example first exon of prolan a β (FSH β) gene), then needn't have or better deletes external source ATG.

Can obtain from natural origin, or use genetic engineering technique or synthetic method to produce the DNA of construct.

Target gene and products therefrom

After in cell such as elementary, secondary or permanence cell are arrived in transfection, DNA construct is the expression of the active or functional part of the required product of may command such as protein or RNA.Product for example can be hormone, cell activator, antigen, antibody, enzyme, thrombin, transport protein matter, acceptor, adjusting albumen, structural protein, transcription factor, sense-rna or ribozyme (ribozyme).In addition, product also can be non-existent protein of nature or nucleic acid (being syzygy protein or nucleic acid).

Method as herein described can produce one or more treatment products, as erythropoietin, thyrocalcitonin, tethelin, Regular Insulin, insulinotropin, insulin-like growth factor, Rat parathyroid hormone 1-34, interferon beta and nerve growth factor, FSH β, TGF-β, tumour necrosis factor, hyperglycemic-glycogenolytic factor, bone growth factor 2, bone growth factor 7, TSH-β, interleukin-11, interleukin-22, interleukin-13, interleukin 6, interleukin 11, interleukin 12, the CSF-granulocyte, the CSF-scavenger cell, CSF one granulocyte/scavenger cell, immunoglobulin (Ig), catalytic antibody, protein kinase C, grape sugar cerebrosidase, the superoxide dismutase, tissue plasminogen activator, urokinase, Antithrombin III, DNAse, alpha-galactosidase, tyrosine hydroxylase, factor V, proconvertin, blood coagulation factor VIII, plasma thromboplastin component, factor X, Hageman factor I, apolipoprotein E or apolipoprotein A-I, globin, low density lipoprotein receptor, the IL-2 acceptor, the IL-2 antagonist, α-1 antitrypsin, immune response modifier and solubility CD4.

But selection marker and amplification

Can use one or more selectable marker gene to be beneficial to identify the target process of hitting.These signs can be included in and hit in the target construct or appear on the different constructs.But selection marker can be divided into two classes: positive selectable and negative selectable (in other words, promptly carrying out positive the selection or the negative sign of selecting).In the positive is selected, (CAD, aspartate transcarbamylase and dihydro whey enzyme, glutamine synthetase (GS), multi-drug resistance 1 (mdrl) and Histidine D (his D) survival as neo, xanthine one guanine phosphoribosyltransferase (gpt), dhfr, adenosine deaminase (ada), tetracycline (pac), Totomycin (hyg), encoding carbamoyl phosphate synthetase are handled, and wherein hit the target construct and have been incorporated into the interior cell of host cell gene group thereby can select but the cell of expressing positive selection marker can enough selective agents.In feminine gender is selected, but the cell of expression feminine gender selection marker is destroyed in the presence of selective agent.Can use one or more marker gene that present negative selectivity characteristic to be beneficial to identify the target process of hitting, but the feminine gender selection marker is connected on the foreign DNA, but but form arrange the relation be to make the feminine gender selection marker be connected the flank that hits target sequence, but thereby and with the host cell gene group in sequence form the stable integration (Mansour that correct homologous recombination can not cause the feminine gender selection marker, S.L.et al., Nature 336:348-352 (1988)).The sign that is used for this purpose comprises herpes simplex virus thymidine kinase (TK) gene or bacterium gpt gene.

But can be incorporated into various selection markers elementary, secondary or the permanence cell in.For example, but can use to have and can select phenotype such as drug resistance, auxotroph, the resistance of pair cell toxic agents or the selection markers such as expression of surface protein.But spendable selected marker gene comprises neo, gpt, dhfr, ada, pac, hyg, CAD, GS, mdr1 and hhisD.The phenotype selected of being given makes to be had the possibility evaluation and separates recipient cell.

But the amplifiable gene (as ada, GS, dhfe and multi-functional CAD gene) of coding selection marker has the feature of adding, but this feature can make them can select to contain the cell of the copy that increases of the selection marker that is inserted in the genome.This feature provides a kind of mechanism of copy number of adjacent or continuous gene of remarkable increase expectation amplification.But also can use his extension increasing sequence of the mutant that shows these sequences improved selectivity characteristic and other.

The ordering of each component part can be different in the DNA construct.When construct was circular plasmids, the order of each element can be in the resulting structures: hit target sequence-plasmid DNA (constituting) by being used for selecting and/or duplicate the sequence that microorganism or other suitable hosts hit the target plasmid but-selection marker-adjusting sequence-exon-splice donor site.Be to use the restriction enzyme cutting of cutting one or many in hitting target sequence to contain the plasmid that hits target sequence and foreign DNA element preferably, linear or the notched molecule with generation before importing recipient cell, thus make the terminal frequency that increases expection homologous recombination process as described herein of dissociative DNA.In addition, available exonuclease handle the dissociative DNA end with generation dangle outstanding 5 ' or 3 of single-stranded dna end ', thereby increase the frequency of expection homologous recombination process.In this embodiment, the homologous recombination of hitting between target sequence and the cellular targets will produce the copy that hit target sequence of two side joints on being included in the element that is imported in the plasmid.

As construct is linear, and this subsequence is as being: but first hit target sequence-selection marker-adjusting sequence-exon-splice donor site-second and hit target sequence; Perhaps can be but that first DNA-second that hits target sequence-adjusting sequence-exon-splice donor site-coding selection marker hits target sequence.Stably the cell of construction and integration body will be handled with the selective agent survival; A subgroup of the cell of stable transfection will be the homologous recombination somatocyte.Available PCR, Southern hybridization and phenotypic screen multiple technologies are identified the homologous recombination somatocyte.

In another embodiment, the order of construct each several part can be: but first hit target sequence-selection marker-adjusting sequence-exon-splice donor site-intron-acceptor splicing site site-second and hit target sequence.

Perhaps, the subsequence of each integral part is as being in the DNA construct: but but first hit target sequence-selection marker 1-and regulate sequence-exon-splice donor site-second and hit target sequence-selection marker 2, but but perhaps can be first to hit target sequence-adjustings sequence-exon-splicing donor confession point-selection marker 1-second and hit target sequence-selection marker 2.In this embodiment, but selection marker 2 performance has the negative feature of selecting.In other words, can according to not contain certain reagent (generally being medicine or metabolite analogue) but the appropriate culture medium formulation in growth select the gene product of selection marker 2, but wherein said reagent can kill the cell of expression selection marker 2.But but side joint selection marker 1 hit the positioning integration that the reorganization between homologous sequence in target sequence and the host cell gene group causes selection marker 1, but selection marker 2 then is not integrated.But but such regrouping process produce by selection marker 1 stable transfection but not by the cell of selection marker 2 stable transfections, but but and can be according at the selective agent that contains selection selection marker 1 and do not select the growing state in the substratum of selective agent of selection marker 2 select these cells.

But DNA construct also can comprise the positive selection marker of the cell of the copy that increases that allows selection to contain this sign.Such sign that increases causes the coamplification of side joint dna sequence dna.In this embodiment, the ordering of each component of construct for example is: but but first positive selection marker-second selection marker (the optional)-adjusting sequence-exon-splice donor site-second that hits target sequence-can increase hits the target DNA sequence.

In this embodiment, but can be by comprising the further gene that is activated of amplification of selected marker gene, but described marker gene has the character of cell that can select to contain the copy that increases of selected marker gene by culturing cell in the substratum that suitably can select preparation is arranged.But the native gene that is activated will be amplified in succession with the selected marker gene that has increased.The cell that contains the copy of many native genes that are activated can produce the very desired protein of a large amount, and can be used for external protein production and gene therapy.

In any embodiment, can select and can increase marker gene all needn't be directly adjacent to each other.

Randomly but DNA construct can contain bacterium replication origin and cell antibiotics resistance sign or other selection markers of permission a large amount of propagation plasmids in bacterium or any other suitable clone/host system.But can use the DNA that comprises the coding selection marker together with the DNA construct at appended sequence such as promotor and splicing juncture to give transfected cell can select phenotype (as the plasmid pCDNEO that schematically illustrates among Fig. 4).Can use methods described herein that such DNA construct is arrived in elementary or the secondary cell together with hitting target DNA sequence cotransfection.

Transfection and homologous recombination

According to present method, as in unique DNA construct or separated DNA sequence transfered cell such as elementary, secondary or the permanence cell, wherein the separated DNA sequence is incorporated in the karyomit(e) or nuclear DNA of transfected cell gradually with construct.

But can will contain at the unique DNA construct or on isolating construct hit target sequence, regulate sequence, the hitting in the target DNA construct transfered cell of exon, splice donor site and selected marker gene.The total length of DNA construct will be decided according to the number of component (but routine forward play target sequence, adjusting sequence, exon selected marker gene and its element of base) and the length of each component.The length of whole construct is generally at least about 200 Nucleotide.In addition, this DNA can be used as line style, bifilar (being with or without the sub-thread district at an end or two ends), sub-thread or cyclic DNA and is imported into.

Then that the construct transfered cell of any form disclosed by the invention is interior to obtain transfected cell.Under the condition that allows the generation homologous recombination as known in the art, keep transfected cell (Capecchi, M.R., Science 244:1288-1292 (1989).When keeping the homologous recombination cell under the condition that is being enough to transcription DNA, the regulatory region (as promotor) that is hit the importing of target construct is with the activated transcription process.

Can be by multiple physics or chemical process with in the DNA construct transfered cell, these methods comprise the transfection of electroporation, microinjection, micropellet bombardment, calcium phosphate precipitation and liposome, polyethylene (polybrene-) or deae dextran mediation.Also can use relevant parotitis of infectivity carrier such as retrovirus, simplexvirus, adenovirus, adenovirus and poliovirus carrier to import DNA in addition.

Randomly also can will hit in the target DNA transfered cell by the segmental form of two or more separated DNA.Using under two segmental situations, one segmental 3 ' terminal and another is segmental 5 ' terminally share dna sequence dna homology (overlapping), and fragment is carried first and is hit target sequence and another and carry second and hit target sequence.When transfered cell, two fragments can be carried out homologous recombination to form single fragment, wherein first and second overlapping regions that hit between target sequence side joint two original segments.The products therefrom fragment is the homologous recombination that is applicable to the cellular targets sequence like this.Also can use plural fragment, design will make these fragments can carry out homologous recombination each other, is suitable for as stated above product with the reorganization of cellular targets sequence homology with last formation.

The homologous recombination somatocyte

Hit the target process and cause hitting the insertion of the adjusting sequence of target construct, native gene is under their control (for example inserting promotor or enhanser by the upstream at native gene or regulatory region).Randomly, hit the disappearance that the target process can cause endogenous regulatory element simultaneously, as the disappearance of the negative regulatory element of tissue specificity.Hit the replaceable existing element of target process, for example tissue-specific enhancer can be had wideer than natural element or different cell type-specific enhanser replaces, and perhaps presenting has adjusting or the induced character that is different from corresponding non-transfected cells.In this embodiment, naturally occurring sequence is deleted and added new sequence.In addition, also can be that endogenous regulatory element is not removed or replaces, but make it to destroy or lose activity as the target process of hitting at exogenous array in the endogenous regulatory element by hitting the target process.

After with the DNA transfered cell, known in the art, be suitable under the condition that homologous recombination takes place between genomic dna and the DNA that is introduced into part, keeping cell (Capecchi, M.R., Science 244:1288-1292 (1989)).

Homologous recombination between genomic dna and the DNA that is imported into produces homology recombinant cell such as fungi, plant or zooblast, particularly elementary, secondary or permanence people or other mammalian cell, the sequence that wherein changes the native gene expression is operably connected on the native gene of certain product of coding, produces to have expression that is different from native gene and/or the new transcription unit of encoding potential.Specifically, the present invention includes and contain the homologous recombination somatocyte of regulating sequence and exon, said adjusting sequence and exon side joint have splice donor site, import and are operably connected on second exon of native gene at predetermined site by hitting the target DNA construct.Randomly, external source exon (coding or noncoding) and intron on a plurality of any exons that are operably connected to native gene also can be arranged.Selecting the amplification coding resulting homologous recombination somatocyte of cultivation under the condition of DNA of sign and new transcription unit's (as suitable) that can increase.No matter whether increase, all can under the condition that is suitable for protein expression known in the art, cultivate cell, thereby, perhaps cell is used for delivering therapeutic protein in the body (being gene therapy) at produced in vitro protein with this method generation.

The said primary cell of this paper comprise be present in from vertebrates tissue source isolated cells suspension (with them auxilliary apply be combined in tissue culture such as plate or culturing bottle shortly before) cell, be present in cell (these two kinds of cells all are to be used for the prototype cell of bed board for the first time) in the explant of self-organization and the cell suspension that obtains from these bed board cells.Term secondary cell or cell strain be meant in cultivation all continue after the cell of step.In other words, from culture medium, remove the primary cell of bed board for the first time and (the going down to posterity) of bed board again, and continue after all cells this paper of going down to posterity be called secondary cell.Secondary cell is the cell strain that the secondary cell by the one or many that goes down to posterity constitutes.A kind of cell strain that is made of secondary cell is: one or many 1) has gone down to posterity; 2) in culture, have the average population multiplication of limited number characteristic; 3) performance have contact inhibition, anchorage dependence growth characteristics (anchorage dependence also is not suitable for value-added cell in suspension culture); And 4) non-ly change forever.

The permanence cell is clone (fixed " cell strain " this definition is relative with aiming at the primary and secondary cell), and its principal character is that they show the obviously unlimited life-span in culture.

The cell of selecting for use for this subject methods can be divided into four types or big class: the cell of not making or contain protein or product (as the fused protein that is not had usually by the protein of cell expressing or nature usually) during 1) as former obtaining; 2) make or contain the cell of certain protein or product, but its manufacturing or amount are not desired amount (be lower than as its amount on the physiology of its archeocyte when resultant common low-level); The common low-level manufacturing protein or the cell of product on one physiology of the archeocyte during 3) as former obtaining, but its content or output will be enhanced or increase; And 4) be expected to change wherein the encode adjusting of certain proteinic gene or the cell of induced character.

Can obtain using elementary, the secondary and permanence cell of present method transfection from various tissues, and comprise the cell type that all can be kept in culture.For example, the primary and secondary cell of available present method transfection comprises formation composition (as lymphocyte, medullary cell), myocyte and these somatic precursor cells of inoblast, keratinocyte, epithelial cell (as mammary epithelial cell, intestinal epithelial cell), endotheliocyte, spongiocyte, neurocyte, blood.Will be used for gene therapy as the homology recombinant cell, primary cell better is to obtain in the individual body that will accept transfected elementary or secondary cell.But primary cell can obtain from donor of the same race (individuality beyond the acceptor).

Also can produce homologous recombination body permanence cell and be used for protein production or gene therapy with present method.Be used for comprising but be not only limited to HT1080 cell (ATCC CCL 121) by the example that present method is produced the permanence human cell line of protein or gene therapy, the derivative of HeLa cell and HeLa cell (ATCC CCL 2,2.1 and 2.2), MCF-7 breast cancer cell (ATCC BTH 22), K-562 leukemia cell (ATCC CCL 243), KB cancer cells (ATCC CCL 17), 2780AD ovarian cancer cell (Van der Blick, A.M.et al., Cancer Res., 48:5927-5932 (1988)), Raji cell (ATCC CCL 86), Jurkat cell (ATCC TIB 152), Namalwa cell (ATCC CRL 1432), HL-60 cell (ATCC CCL 240), Daudi cell (ATCC CCL 213), RPMI 8226 cells (ATCC CCL 155), V-937 cell (ATCC CRL 1593), Bowes melanoma cells (ATCC CRL 9607), WI-38VA13 subbreed 2R4 cell (ATCC CCL 75.1) and MO LT-4 cell (ATCC CRL1582), and through merging the xenogenesis hybridoma that people's cell and another kind cell are produced.Can use secondary human fibroblasts's cell strain such as WI-38 (ATCC CCL 75) and MRC-5 (ATCC CCL 171).In addition, can use elementary, secondary and permanence people cell, and elementary, the secondary or permanence cell that demonstrates other kind of outer-gene amplification character carry out external protein production or gene therapy.

Genetic modification is become the method for cDNA copy

The invention still further relates to and use homologous recombination genetic modification to be become the method for cDNA copy (gene copy that does not have intron).CDNA copy can be transformed in yeast or the bacterium to carry out external protein production, perhaps the cDNA copy be inserted in the mammalian cell to carry out external or the production of body internal protein.If cDNA will be transferred in the microorganism cells, can import two through homologous recombination and contain the DNA construct of hitting target sequence, one is the upstream construct of the people's gene of coding therapeutic protein, one is the downstream construct of said gene.For example, the sequence that imports the upstream comprises the DNA that starts microorganism cells excretory leading peptide with the coding that repeats (LTR) for a long time, is coded in the sequence that is used for the sign selected in the microorganism cells, the regulatory element of function is arranged and have a splice donor site at microorganism cells through first amino acid whose DNA of the therapeutic protein of processing or its upstream gene group dna sequence dna homologous dna sequence dna, retrovirus of encoding mature.The sequence that imports the upstream is near the first amino acid whose genomic dna of the therapeutic protein through processing of encoding mature and the upstream is imported into.The sequence that imports the downstream comprise with encoding mature through proteinic last amino acid whose DNA of processing or the genomic dna sequence homologous dna sequence dna in its downstream, the transcription termination sequence of microorganism, the sequence and the retrovirus LTR that can instruct DNA in microorganism cells, to duplicate.The sequence that imports the downstream is to be imported in the DNA adjacent and the downstream of the terminator codon of the therapeutic protein through processing of encoding mature.After these two DNA construct are imported in the cell, under the condition that is suitable for being imported into generation homologous recombination between DNA and genomic dna, keep the gained cell, thereby produce the homologous recombination somatocyte.Sometimes, one of DNA construct or both codifieds are used to contain one or more signs that the positive or negative of the cell of DNA construct is selected, and can select step with increasing by one in the method behind one of them or the two kinds of DNA construct transfered cells.In addition, the sequence that is coded in the sequence of the selection marker in the microorganism cells and can instructs DNA to duplicate in microorganism cells can be that the both is present in the upstream or down in the guerrilla warfare target construct, perhaps can be that the sign that is used for selecting in microorganism cells is present in guerrilla warfare target construct down and the sequence that can instruct DNA to duplicate may reside in the guerrilla warfare target construct in microorganism cells.Under the condition of the processing of the RNA product of the gene that is suitable for the therapeutic protein of transcribing, encode that LTR instructs and reverse transcription, cultivate the homologous recombination somatocyte then.The product of reverse transcription is the DNA construct that contains the intronless DNA copy of the therapeutic protein of encoding.It is operably connected on the dna sequence dna that contains above-mentioned two foreign DNA constructs.To import in the microorganism cells by the intronless DNA construct that present method is produced then.Under expression that is suitable for therapeutic protein and excretory condition, cultivate this microorganism cells.

The body internal protein is produced

Homologous recombination somatocyte of the present invention can be used as colony, the colony as the elementary secondary cell of homologous recombination body, homologous recombination body clonal cell line or clone, homologous recombination body allos cell strain or the clone of homologous recombination somatocyte system, and uses as the cell mixture of at least a representative cell that wherein contains one of aforementioned four class homology recombinant cells.Such cell can be used for treating suffering from the therapeutic product is transmitted unusually or the not transfer system of the individuality of expectation state react, and said therapeutic product is: 1) therapeutic protein (do not have in for example individual body, under production with respect to the physiological requirements of individuality, individual defective or ineffectually or the protein that utilizes inadequately; The protein that new function such as enzymatic or transportation function are arranged), or 2) therapeutic nucleic acids (as inhibition of gene expression or have the active RNA of intrinsic enzymatic).In the method that therapeutic protein or nucleic acid is provided of the present invention; give suffer to be treated or prevention unusually or the individuality of the pathological state of not expecting; with homologous recombination body primary cell, clonal cell line or the allos cell strain of suitable approach use capacity, with this protein or the foreign DNA of on the physiology related levels, expressing or preparation can get.The physiology related levels is near the normal product level that produces in the body or causes unusually or product level that the state of not expecting is improved.According to embodiment of the present invention as herein described, be ready to use in individual homologous recombination body permanence clone and can be changed in one or more semipermeable barrier devices.The penetration property of device is can stop the cell separating device after will be in implanting animal body, and the therapeutic product can freely see through and also can leave barrier device and enter the local space around the implant or enter systemic circulation.For example, hGH, hEPO, people's insulinotropin, hGH-CSF, hG-CSF, human alpha interferon or people FSH β whole body in human body are phoresied and help the treatment.

Barrier device is useful especially, and can make the homologous recombination body permanence cell that will implant, from the homologous recombination somatocyte (the external source cell of homologous recombination) of other kind, or mate the pathological state of the cell (homologous recombination body homogeneous variant cell) of donor from non-histocompatibility, or on agricultural, use (promptly being used for meat and milk production) in order to the treatment human or animal.When because any former thereby will stop to treat the time, barrier device can be by providing promptly near carry out short-term (promptly of short duration) treatment easily with the approach of removing cell.

There are many synthetic, semisynthetic or natural filter membrane all to can be used for this purpose, they comprise, but be not limited to the polymkeric substance of Mierocrystalline cellulose, cellulose acetate, nitrocellulose, polysulfones, poly(vinylidene fluoride), polyvinyl chloride polymer and polyvinyl chloride derivative.When utilizing barrier device, can make the gene therapy that also can be used for the people from elementary, the secondary or permanence cell of another kind.

External protein production

According to the present invention, also can be used for external protein production from the homologous recombination somatocyte of people or inhuman kind.Under condition known in the art, as to cause protein expression, keep cell.Be the required protein of purifying, can use stated method with expressed protein with purifying in cellular lysate or the cell conditioned medium liquid.The protein that makes according to present method comprises therapeutic protein, its available conventional route of administration known in the art (as in oral, intravenously, intramuscular, the nose or approach such as subcutaneous) be transported in people or the non-human animal's body.These protein comprise hGH, hEPO and people's insulinotropin, hGM-CSF, hG-CSF, FSH β or interferon-alpha.These cells can be permanence, elementary or secondary cell.When inhuman cell helps carrying out therapeutic or commercial non-human protein's protein production, can use cell from other kind, for example use the cell that derives from salmon to produce salmon calcitonin see calcimar, use derives from the cell of pig with the production pork insulin, and uses the ox cell to produce Trobest.

In another embodiment, the invention describes the method that in transfected elementary, secondary or permanence cell, activates the native gene of (promptly opening) and the required product of amplification coding.Promptly the present invention relates to the method that imports through with the genomic dna homologous recombination, dna sequence dna is not connected with the native gene function usually, and (1) when inserting it in host genome on native gene or near it, be used for changing the expression of (as activating) native gene, and also have (2) to allow to select those wherein cells of being amplified of activated native gene.But being used for DNA amplification sequence of the present invention comprises, but be not limited to, but the sequence and the CAD gene (encode three functional protein carbamyl phosphate synthase, aspartate transcarbamylase and dihydroorotase) of coding selection marker Tetrahydrofolate dehydrogenase, adenosine deaminase.But also can use the variant of the improvement of these sequences and other extension increasing sequence.The method according to this invention, but but with the DNA amplification sequence of coding selection marker and the dna sequence dna that changes the regulatory function that native gene expresses the preselected site of cellular genome import elementary, secondary in the mode that interrelates with genomic dna homologous dna sequence dna or the permanence cell in.This preselected site generally is within the gene of coding therapeutic product or its upstream, or on the site of the required gene function of an influence.Can be used as the unique DNA construct, or as physically becoming the separated DNA sequence that is connected transfected gene expression of cells, but but with change dna sequence dna that native gene expresses, coding selection marker extension increasing sequence and with genomic dna in preselected site homologous sequence import in elementary, secondary or the permanence cell.In addition, can be linear, the distrand DNA that is with or without the sub-thread zone at an one end or two ends imports DNA, or with annular DNA DNA imported.After with the foreign DNA transfered cell, under the condition that homologous recombination takes place between the part of the DNA that is suitable for genomic dna and is imported into, keep this cell.Genomic dna and the homologous recombination that is imported between the DNA cause elementary, the secondary or permanence cell of homologous recombination body, but but sequence that the change native gene is expressed in these cells and coding selection marker extension increasing sequence all are operably connected on the native gene of coding therapeutic product.But under the amplification condition of the DNA amplification of selecting the coding selection marker, cultivate the homology group group somatocyte of gained, but produce the cell of the native gene that contains selection marker that has increased and the coamplification that has changed its expression.Can under the condition of the expression that is suitable for therapeutic protein, cultivate the cell that produces with this method, thereby, maybe cell can be used for transmit in the body therapeutic protein (being gene therapy) at external generation therapeutic protein.

The invention still further relates to the method in the genomic dna that will hit target DNA sequence transfered cell, wherein construct comprises the foreign DNA of coded product, the marker gene of hitting target sequence and can increase, after like this in being incorporated into the genome of cell, cell contains the DNA of the coding maker that has increased and the foreign gene of coamplification, and wherein these cells can be expressed the foreign gene of coamplification.

But DNA construct comprises positive selection marker, and it can be used for selecting to contain the cell that increases and copy of this sign.Such sign that increases causes the coamplification of side joint dna sequence dna.In this embodiment, the order formed of construct for example can be: but but the DNA-that hits DNA-second selection marker of coding (if any) of the positive selection marker that target sequence-coding can increase be equivalent under suitable promotor control, to treat expressed exogenous gene or in the dna sequence dna that has only its location with the promotor of activation native gene-hit target DNA sequence.

Advantage

Methodology of the present invention, DNA construct, cell and gained protein have many-sided function and surpass many other advantages that gene hits present method therefor in the target technology.By from the useful gene of direct neighbor (directly merging mutually) to native gene with the transcriptional domain of normal gene by transcriptional domain upstream 30kb or upstream end more; or the location of the different positions in the intron of native gene external source adjusting sequence, be particularly advantageous for the ability that activates native gene aspect the intracellular genetic expression.For example, it can be used for being positioned at usually regulatory element static or gene being had the upstream or the downstream in the zone of negative regulatory function.Upstream or downstream location regulatory element in such zone can be eliminated the main negative effects that this common inhibition is transcribed.In addition, can use the DNA zone that the deletion of target construct suppresses to transcribe or expression of gene is had other disadvantageous effect usually of hitting as herein described.

In addition, because known promoter function depends on local environment to a great extent, so be suitable for the local environment of function performance and the location that can grope wide region in order to find out those.Yet, because of the ATG initiator codon sees (approximately per 48 base pairs occur 1) in the mammalian DNA continually, so any position of upstream region of gene simply transcriptional start and be created in correct ATG initiator codon before contain the transcript of long preambles sequence will hinder translating of correct gene product and information was lost efficacy because the ATG codon in such leader sequence, frequently occurs.Therefore, mix external source exon, splice donor site and can be randomly in the target construct containing hitting of regulatory region, a kind of intron and acceptor splicing site site, can allow to be tested and appraised that optimized site reaches the optimum expression of gene for regulation domain, and needn't be subjected to avoid showing the restriction that unsuitable ATG initiator codon is applied at the mRNA that is produced.So just obvious greater flexibility is provided and has made it to activate the more gene of wide region for the location construct.DNA construct of the present invention also is useful in the syzygy method of protein of making by recombinant chou or exogenous array and endogenous sequence coding for example.

Gene mentioned above hits target and is specially adapted to change the expression of gene (said transcription unit is sufficiently greatly to cause them to be difficult to separate and express) that forms transcription unit with amplification, or is applicable to and opens that those wherein complete protein coding regions can not obtain or as yet not by cloned genes.Therefore, above-mentioned DNA construct is applicable in a certain way the external source regulatory element is operably connected on the native gene, said mode is accurately to limit transcription unit, for the relative positioning of external source regulatory element and native gene provides handiness, finally enable to become one obtain and adjustment of treatment on the system of height control of valuable genetic expression.

Explanation to embodiment

As described herein, the applicant has proved and can will also can be incorporated in the genome of transfected cell through homologous recombination in DNA transfered cell such as elementary, secondary or the permanence vertebrate cells.They show that further foreign DNA has the function of expection in homologous recombination body (HR) cell, and can identify correct target cell based on the detected phenotype of being given by suitable target DNA.

The applicant describes in order to the structure of the plasmid that hits particular seat in the people's gene group (HPRT seat) with based on the selection (embodiment 1a) of drug resistance phenotype.This plasmid is named is pE3Neo, and it is incorporated at the HPRT seat to produce in the cellular genome has hprt ^-, 6-TG resistant phenotype and also have the cell of G418 resistance.As mentioned above, by proof, be imported into the location of DNA in the exon 3 of hprt gene in the clone set up after, they have shown and have hit at gene that pE3Neo can suitably bring into play function (embodiment 1b) in the target in the human fibroblast cell line who has set up.

In addition, applicant's proof can be used pE3Neo to carry out gene in the primary and secondary human skin fibroblast and hit target (embodiment 1c).The application proves that further the modification to the DNA end can improve the hit rate (embodiment 1c and 1e) that DNA enters genomic dna.The applicant has also described and has hit target technology with gene and gene is inserted method (embodiment 1d) in the genome of cell such as elementary, secondary or permanence cell in the site of preliminary election.

In addition, the present invention relates to use transfected cells produce method of protein.This method comprises with the foreign DNA of coding treatment product or with being enough to hit to coding treats the DNA of endogenous gene of product and transfectional cell, as elementary cell, secondary cell or permanence cell.For example, embodiment 1g, 1h, 1j, 1k, 2,3,4 and 6-9 described by the endogenous gene of selecting being hit target with the production method of protein with changing the dna sequence dna element that native gene expresses.

The applicant has also described to be used to increase and has hit the DNA construct and the method (embodiment 3,6,8 and 9) of target activated endogenous cellular gene by gene.

Embodiment 1f～1h, 2,4 and 6 has described embodiment of the present invention, wherein change the upstream normal regulating sequence of people EPO gene, express hEPO to allow under not transfected state to reach in the elementary or secondary inoblast strain of EPO with detectable scale.In one embodiment, the product that hits target has kept normal epo protein matter intact just, but must be under the control of mouse metal sulfur hydrochloride promotor.Embodiment 1i and 1j proof can be used and similar hit the target construct to activate the endogenous growth hormone gene among the elementary or secondary human fibroblasts.The EPO that has described in other embodiments to activating among the human fibroblasts expresses, and the product that hits the target process is chimeric transcription unit, and wherein first exon of human growth hormone gene is positioned in the upstream of EPO exon 2～5.The product of transcribing (controlled by the mouse metal sulfur hydrochloride promotor), splice and translating is a protein, and wherein the amino acid/11 of hEPO signal peptide～4 are replaced by the amino-acid residue 1-3 of hGH.This proteinic telescoping part, signal peptide are being removed before the secretion from cell.Embodiment 5 has described the expressed cDNA copy (intronless) that gene (intron is arranged) is changed into this gene in order to produce, and reclaim in microorganism (as yeast or bacterium) cell that these can express the cDNA molecule hit target construct and method.Embodiment 6 has described cell that wherein hhfr gene has been amplified and has carried out double selection and selection, names the structure into the hit target carrier of pREPO4.Plasmid pREPO4 has been used to people EPO (hEPO) seat that increases in HT 1080 cells (a kind of permanence human cell line) after activating endogenous hEPO gene by homologous recombination.As previously mentioned, substep is selected to make and is had the hEPO turnout in the cell of resistance to increase by 70 times to 0.4 μ M methotrexate in containing the methotrexate substratum.

Embodiment 7 and 8 has described in the upstream of normal EPO promotor and has inserted the method for regulating sequence and use such construct to produce the method for EPO.In addition, embodiment 8 has described the method for amplification by the target EPO gene of the method production of embodiment 7.Embodiment 9 has described target human alpha interferon, GM-CSF, G-CSF and FSH β gene are used for the cell of protein production with generation the method for hitting.

Embodiment provides by gene and has hit the method for target with activation or activation and amplification native gene, does not wherein need to operate or use in addition the protein coding region of target gene.Use method and DNA construct or plasmid or its conspicuous for those of ordinary skills modified forms of this paper instruction; can change the characteristic (as producing medicine) that expectation has external protein production, or have the genetic expression in the cell of the feature that is suitable for body internal protein transfer approach (as gene therapy).Two kinds of schemes that activate the hEPO gene are transcribed in Fig. 5 and 6 graphic extensions.

Use the conspicuous to those skilled in the art modified forms of method disclosed herein and DNA construct or plasmid or its, the foreign DNA of therapeutic product (as protein, ribozyme, sense-rna) of can will encoding in the site of pre-selected inserts in the gene cell of the elementary or secondary cell of vertebrates (as people and non-human mammal).

Now the present invention is described by the following example, but these embodiment and not limiting the present invention in any way.

Embodiment

Embodiment 1 hits target by gene and produces transfected cell strain

Producer hits target when by homologous recombination transfection DNA being incorporated into or partly replace chromosomal DNA sequence.Though this situation may occur in the process of any specific transfection experiment, they are usually by too much being covered up by the caused plasmid DNA integration of nonhomologous or irrational regrouping process.

A. be used for the generation of selecting people's cell gene to hit the construct of target

A kind of method that the target consequence is hit in selection is to select because the gene function that integration the caused forfeiture of transfection DNA by heredity.People HPRT seat coding xanthoglobulin-phosphoribosyl transferase.Can select hprt according to growing state in the substratum that contains nucleoside analog 6-sulfenyl guanine (6-TG) ^-Cell: have the allelic cell of wild-type (HPRT+) and killed, and have mutant (hprt by 6-TG ^-) allelic cell can survive.Therefore can in the 6-TG substratum, select to contain the cell of the target that destroys the hprt gene function.

In order to make up the plasmid that hits the HPRT seat, with HPRT sequence (Genedank title HUMHPRTB; Edwards, A.et al., Genomics 6:593-608 (1990)) in from the position 11,960～18,869 exon 2s that extend and that comprise hprt gene and 3 6.9 kbHindIII fragment subclones in the HindIII site of pUC12.Cutting institute's DCRP and insertion contain 1.1 kb SalI-XhoI fragments from the neo gene of pMClNeo (Stratagene) on unique XhoI site in the segmental exon 3 of hprt gene, destroy the encoding sequence of exon 3.Select one to have directed person and the called after pE3Neo that transcribes opposite neo transcriptional orientation with HPRT.Replace normal HPRT exon 3 with neo destructive variant and will produce hprt ^-, the 6-TG resistant phenotype.Such cell also will be the G418 resistance.

B. the gene in the human fibroblast cell line who has set up hits target

As hitting the evidence of target in permanence clone, and determining to hit at gene that pE3Neo suitably brings into play function in the target, is HT 1080 (ATCC CCL 121) by electroporation with pE3Neo transfection human fibrosarcoma cell.

Before electroporation, HT 1080 cells are maintained and be added with the containing among the 15% calf serum DMEM (Hyclone) of HAT (xanthoglobulin/aminopterin/xanthine).Electroporation a few days ago with cell transfer in the same substratum of no aminopterin.Dilute with the trysinization index growthing cell and in the DMEM/15% calf serum, centrifugal, and with every milliliter 13.3 * 10 ⁶The cell final volume of cell is suspended among the PBS (phosphate-buffered saline) again.Digest pENeo with HindIII, 8 kb HPRT-neo fragments are separated with PUC 12 main chains, carry out purifying through the ethanol sedimentation of phenol extraction, and suspend again with the concentration of 600 μ g/ml.Get 50 ml (30 μ g) together with 750 μ l cell suspensions (10 * 10 ⁶Cell) is added in the electroporation Xiao Chi (0.4cm interelectrode distance, Bio-Rad Laboratories).Electroporation conditions is 450 volts, 250 microfarads (Bio-Rad Gene Pulser; Bio-Rad Laboratories).The Xiao Chi content is added to immediately obtains every 25ml substratum among the DMEM that contains 15% calf serum and contain 1 * 10 ⁶The cell suspension of cell.The cell suspension shop that 25ml is treated is applied in 150mm diameter tissue culture dish and at 37 ℃ and is contained 5%CO ₂Be incubated in the environment.After 24 hours, G418 solution directly is added on the plate, obtains the G418 that final concentration is 800 μ g/ml.After 5 days, replace substratum with DMEM/15% calf serum/800 μ g/ml G418.Behind the electroporation 9 days, replace substratum with DMEM/15% calf serum/800 μ g/ml G418 and 10 μ m 6-sulfenyl guanines.The colony that used the collection of clone's cylinder G418 and 6-TG to be had resistance in 14～16 days after the beginning double selection.

5 representative target result of experiment of hitting of carrying out in HT 1080 cells are shown in Table 1.

The number G418 of cell is handled in table 1 transfection ^rAnd 6-TG ^r

Clone's number 11 * 10 ⁷322 1 * 10 ⁷283 1 * 10 ⁷244 1 * 10 ⁷325 1 * 10 ⁷66

For transfection experiment 5, design is to determine G418 ^rThe contrast plate of the total yield of colony shows can be from initial 1 * 10 ⁷Individual processed cell produces 33,700 G418 ^rColony.Therefore, by the person of hitting to being not 66/37,700 or 1: 510 by the person's of hitting ratio.With 5 experiment joint accounts, the person's of hitting frequency is 3.6 * 10 ⁶, or account for 0.00036% of processed cell.

Restriction Enzyme and use Southern hybrid experiment that the probe be derived from neo and hprt gene carries out with the neo assignment of genes gene mapping in the HPRT exon, give the HPRT seat of locating point.

C. gene hits target in the primary and secondary human skin fibroblast

Digest pE3Neo with HindIII, from the pUC12 main chain, separate 8 kb HPRT-neo fragments, and through phenol extraction and ethanol sedimentation purifying.With the concentration of the 2mg/ml DNA that suspends again.With 100 μ g HindIII pE3Neo (50 μ l), under 250 volts and 960 microfarad conditions, be 3 * 10 of 0.5ml to volume ⁶Individual secondary human foreskin fibroblast is carried out electroporation.To altogether 9 * 10 ⁶Individual processed cell carries out the transfection that separates for three times.Processing treatment is also selected the G418 resistant cell.Every 150mm culture dish middle berth applies 500,000 cells and carries out the G418 selection.Select to cultivate after 10 days down, substratum is changed into the human fibroblasts's nutritional medium that contains 400 μ g/ml G418 and 10 μ M 6-TG.Being used in combination two kinds of medicines continues to select 10 days again.The test under microscope plate is to locate the human fibroblasts's colony that two kinds of medicines is all had resistance.G418 ^rT-TG ^rColony partly is per 9 * 10 ⁶The cell of individual processing has 4 colonies.These colonies account for 0.0001% (perhaps 1,000,000/) of all cells that can form colony.G418 is determined in design ^rThe contrast plate of the overall yield of colony shows can be from initial 9 * 10 ⁶The cell of individual processing produces 2,850 G418 ^rColony.Therefore, the ratio to missing that hits is 4/2,850 or 1 to 712.Carry out the Southern hybrid experiment with Restriction Enzyme and the probe that is derived from neo and hprt gene, determine the HPRT seat of the neo assignment of genes gene mapping, and prove that hitting target occurs in these four clonal cell lines to the interior site of being scheduled to of HPRT exon 3.Also by transfection primary cell (1/3.0 * 10 ⁷) separate colony to two kinds of drug resistants.

These pE3Neo hit the target result of experiment and summarize in table 2.Direct transfection or handle the pE3Neo of HindIII digestion before transfection, to produce 5 ' sub-thread protuberance (seeing embodiment 1c) with exonuclease III.Test has the DNA preparation in the sub-thread zone of length from 175 to 930 base pairs.Only use a pE3neo, cut enzyme and Sou-thern hybridization analysis method identifies 1/799 G418 resistance colony (being separated to 24 clones that hit altogether) according to have the neo gene that inserts that hits on the HPRT seat by restricted through HindIII digestion.When using 175 bp protuberances, hit target and be subjected to maximum stimulation (about 10 times of stimulations), 1/80 G418 ^rColony demonstrates for hitting target at the HPRT place the restriction fragment of the effect of detecting (isolating 9 clones that hit altogether).Therefore, use condition as herein described and recombinant DNA construction body, observe the target that hits in the normal people inoblast at an easy rate, and by giving prominence to the target that hits of afterbody with containing sub-thread, press method transfection described in the embodiment 1e, can stimulate total target frequency (the clone's number that hits is the clone's of G418 resistance a sum divided by being stabilized the ground transfection) of hitting.

Table 2

In the human fibroblasts, the HPRT seat is hit target PE3neo and handle each G418 of number of experiments ^rSeveral total HindIII of clone that hit that hit of clone digest 6 1/,799 24175 bp protuberances, 1 1,/80 9350 bp protuberances, 3 1/,117 20930 bp protuberances 1 1/,144 1

D. the generation that is used for the construct in the gene target insertion people's gene group that treatment is useful is hit the application of target with it at gene

Can use wherein adjacent or approach neo gene place and will treat the varient that useful gene inserts the pE3Neo in the HPRT coding region, will treat the specific position in the elementary or secondary cell genome of useful gene site-directed guiding acceptor.Can make up this varient that the hGH gene is hit to the pE3Neo at HPRT seat.

Digest pXGH5 (being illustrated among Fig. 3) with EcoRT, and separate the 4.1 kb fragments that contain the hGH gene and connect mouse metal sulfur hydrochloride (mMT) promotor.Be used for filling the EcoRI protuberance from the Klenow of intestinal bacteria archaeal dna polymerase fragment.Be used in the XhoI digestion pE3Neo that the joint (insert 3 ' joint in exon 3) of neo fragment and HPRT exon 3 cuts individually.Be used for XhoI protruding terminus from the Klenow of intestinal bacteria archaeal dna polymerase fragment filling linearization plasmid, and the gained fragment is connected on the 4.1 kb blunt end hGH-mMT fragments.The single copy of analyzing on the hGH-mMT fragment with Restriction Enzyme inserts, and is pE3Neo/hGH to screen from ligation mixture deutero-bacterium colony, to select the hGH genetic transcription location identical with neo gene direction and name.Digest pE3Neo/hGH with HindIII, discharge the 12.1 kb fragments that contain HPRT, neo and mMt-hGH sequence.Press described in the embodiment 1c and to handle through the DNA of digestion and transfection in elementary or secondary human fibroblasts.The method described in the embodiment 1c of pressing is selected G418 ^rTG ^rColony is also analyzed mMT-hGH and the decide target of neo sequence in hprt gene inserts.The hGH that commodity in use immunodetection medicine box (Nichols Instit-ute) is analyzed single colony expresses.

With the secondary human fibroblasts of pE3Neo/hGH transfection, the stable hGH that analyzes thioguanine resistance colony expresses, and limiting it property enzyme and Southern hybridization analysis.13 TG that analyzed ^rIn the colony, identifying 8 has the hGH gene to be inserted into colony in the endogenous HPRT seat.All 8 bacterial strains have all stably been expressed the hGH of significant quantity, and average expression level is 22.7 μ g/10 ⁶Cell/24 hour.In addition, also plasmid pE3neo EPO shown in Figure 4 can be used for the EPO gene is decided target guiding people HPRT seat.

Use homologous recombination will treat useful gene and decide lead specific position in the cell genomic dna of target, this technology can enlarge and make it more to be applicable to the product (as medicine, gene therapy) by the production for treating purpose of inserting gene, makes cellular exposure select to contain the cell of this gene copy that has increased under suitable medicament selection envrionment conditions by insert gene in cell.For example, can be in being directly adjacent to pE3neo/hGH the position of hGH or neo gene insert dhfr, ada or CAD gene, thereby modify pE3neo/hGH (embodiment 1d).Elementary, the secondary or permanence cell of available such plasmid transfection also identifies that the correct target of deciding inserts the result.Further with these cells of drug treating (selective agent to dhfr is a methotrexate, and the selective agent of CAD is N-(phosphoric acid ethanoyl)-L-aspartic acid (PALA), is adenosine (as the nitro serine (Ser.)) to the selective agent of ada) of cell that are suitable for selecting to contain amplification gene of cumulative concentration.The integration of useful gene will be able to selecteed gene together with the copy of its amplification by this way by coamplification in the treatment.Therefore, can control the genetic engineering of cell at an easy rate by preselected specific site, with the gene that production for treating is used, hit wherein that the target construct is incorporated in the cellular genome in said site and the copy of amplification in this site is kept at expanded cells.

E. the modifying DNA end hits target efficient with raising

There is the evidence of several respects to show that 3 ' outstanding end participates in some homologous recombination approach of intestinal bacteria, phage, cereuisiae fermentum and Xenopus laevis.In the xenopus oocyte cell, have the molecule that length reaches 3 ' protruding terminus of a hundreds of base pair and more promptly recombinating than the molecule that has through the very short protruding terminus (4 bp) of restriction enzyme digestion back generation after the micro-injection to the molecule of similar processing.In yeast, length reaches the rate-limiting step in the generation reduction division seemingly reorganization of 3 ' protruding terminus of a hundreds of base pair.Still the evidence that is not reported and in the reorganization of people's cell, has 3 ' protruding terminus to participate in, and not expression have the adorned DNA substrate of any kind can be in office which kind of promote to hit the situation of target (a kind of form of homologous recombination) in belonging to.The experiment prompting of describing among the following example and the embodiment 1c, 5 ' outstanding end is effective for stimulating the target that hits among elementary, secondary and the permanence human fibroblasts.

Also do not hit the report of target efficient about improving by the end of modifying the transfection DNA molecule.Present embodiment is intended to illustrate by the end with the molecule of bifilar form and changes the sub-thread form into and the end of linear DNA molecule is modified, and can stimulate that the genomic target that hits inserts in the primary and secondary human fibroblasts.

Digest 1100 μ g plasmid pE3Neo (embodiment 1a) with HindIII.Can directly use this DNA behind phenol extraction and ethanol sedimentation, perhaps available gel electrophoresis is isolated the 8 kb HindIII fragments that only contain HPRT and neo gene from pUC12 carrier sequence.With the DNA that ExoIII digestion digest with HindIII, cause in the circumscribed hydrolytic digestion of nucleic acid widely that originates in each free 3 ' end and produce 5 ' end given prominence to.Therefore can control the degree of the circumscribed digestion of nucleic acid and the length of gained 5 ' overhang by the time that changes ExoIII digestion.The condition of recommending according to supplier digests 30 seconds, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes and 5 minutes with ExoIII with the pE3Neo of 100 μ g HindIII digestion already.In order to monitor the degree of digestion, under the condition that supplier recommends, handle the aliquot sample of the DNA that contains 1 μ g ExoIII processing on each time point with mung bean (mung bean) nuclease (Promega), and through the gel electrophoresis sample separation.The same intermolecular difference in size that detects the pE3Neo untreated, that HindIII digests and handle with ExoIII and mung-bean nuclease.This magnitude difference is divided by 2 mean lengths that obtain each end 5 ' protuberance of molecule.At above-mentioned time point, and in 30 ℃ of digestion, the 5 ' protuberance length range that is produced should be 100 to 1,000 bases.

The DNA (total HindIII digestion product of pE3Neo) that the 60 μ g ExoIII that obtain on each time point of purifying handle, and under the described condition of embodiment 1c with its electroporation in elementary, secondary and permanence human fibroblasts.Calculate G418 ^r6-TG ^rThe number of colony is also compared these numbers with the target that hits that the pE3Neo of the HindIII digestion of not handling with ExoIII carries out, thus the target degree of hitting that the preparation that each ExoIII of quantitative analysis handles improves.

Also can use the effect of the quantitative 3 ' protruding terminus of similar system.In this case, under the condition that supplier recommends, handle the pE3Neo of HindIII digestion at interval with different time with phage t7 gene 6 exonucleases (Vuited Stat-es Biochemicals).Determining of digestion program (mean length of each terminal 3 ' protuberance that produces) is identical with the DNA that above-mentioned ExoIII handles with electroporation conditions.Calculate G418 ^r6-TG ^rThe number of colony is also compared with the colony number that pE3Neo of those HindIII digestion of handling without T7 gene 6 exonucleases hit target, the degree of hitting target that improves with the preparation of quantitatively being handled by each T7 gene 6 exonuclease.

Produce 5 ' and other method of 3 ' protruding terminus be feasible, for example, two linear molecule sex change that partly overlap are each other also annealed and will be produced the mixture of molecule, each molecule has 3 ' protuberance or at two ends 5 ' protuberance is arranged at two ends, and the fragment of annealed again that can not divide with initial linear molecular regime.Determine the length of protuberance according to the length of non-shared DNA between two dna fragmentations.

F. be used for the human erythropoietin gene that is under the control of mouse metal sulfur hydrochloride promotor is put into elementary, secondary and permanence human fibroblasts's the structure that hits the target plasmid

Following embodiment is in order to explanation one embodiment of the invention, wherein the normal positive of human erythropoietin (hEPO) upstream region of gene and the negative sequence of regulating are changed, to allow expressing human erythropoietin in elementary, the secondary or permanence human fibroblasts who does not express hEPO originally with significant quantity.

Available PCR method amplification is positioned at the zone of people EPO upstream of coding region individually.[Genbank names HUMERPA analyzing disclosed people EPO sequence; Lin, F-K, et al., Proc.Natl.Acad.Sci., USA 82:7580-7584 (1985)] three groups of primers that are used for this purpose of back design.These primers are to the fragment of 609,603 or 509 bp that can increase.

Table 3

HUMERPA primer location sequence fragment size F1 2 → 20 5 ' AGCTTCTGGGCTTCCAGAC

(SEQ?ID?NO?1)R2 610→595 5′GGGGTCCCTCAGCGAC 609?bp

(SEQ?ID?NO?2)F2 8→24 5′TGGGCTTCCAGACCCAG

(SEQ?ID?NO?3)R2 610→595 5′GGGGTCCCTCAGCGAC 603?bpF3 21→40 5′CCAGCTACTTTGCGGAACTC

(SEQ?ID?NO?4)R2 610→595 5′GGGGTCCCTCAGCGAC 590?bp

These three fragments overlap basically and can change mutually for this purpose.Will be terminal two with the connection of ClaI linker from-623 609 bp fragments (HUMERPA nucleotide position 2 to 610) that extend to-14 with respect to the rotaring intertranslating start site.Digest the fragment of the ClaI connection of gained with ClaI, and be inserted in the ClaI site of pBluescriptII SK/+ (Stratagene), its direction is to make HUMERPA nucleotide position 610 adjacent with the SalI site in the many linkers of plasmid.Peculiar FspI in the fragment of people EPO upstream or SfiI site (the HUMERPA nucleotide position is respectively 150 and 405) locate to cut this plasmid p5 ' EPO respectively, and are connected on the mouse metal sulfur hydrochloride promotor.Generally the 1.8kb EcoRI-BglII fragment from the mMT-I gene [does not contain the mMT encoding sequence; Hamer, D.H.and WallingM., J.Mol.Appl.Gen.1:273-288 (1982); Also can use the designed PCR primer of mMT sequence that derives from Genbank (being MUSMTI, MUSMTIP, MUSMTIPRM) by analysis, from mouse gene group DNA, separate this fragment by currently known methods] available currently known methods accomplishes tack with it and is connected with (also the accomplishing tack) of SfiI digestion or the p5 ' EPO of FspI digestion.Analyze institute's DCRP direction and will be wherein the former mMT BglII site be used for hitting target near those of SalI site in the many linkers of plasmid and import the primary and secondary human fibroblasts.This future orientation mMT HUMERPA nucleotide position 610 in final construction is transcribed.The resulting plasmid that is used for mMT is inserted FspI and SfiI site is called p5 ' EPO-mMTF and p5 ' EPO-mMTS.

Modify in hope, deletion and/or the negative regulatory element of displacement or be positioned under the situation of enhanser of upstream of primary target sequence, additional upstream sequence is useful.Under the situation of EPO, can delete suppress EPO liver outside and kidney organize outward in the negative regulatory element (Semenza, G.L.et.al., Mol.Cell.Biol.10:930-938 (1990)) of expression.Prepare a series of deletion sequence in the 6 kb fragments.The available zone that the active enhanser of wide host cell (as the enhanser from the huge virus of cell (CMV)) displacement disappearance is arranged.

Because the HUMERPA sequence is after its 5 ' end is positioned at BamHI (far-end) and HindIII (near-end) site, thereby can select the segmental direction of 609bp 5 ' EPO in the pBluescriptIISK/+ carrier.Therefore, available currently known methods separates the 6 bp BamHI-HindIII fragments (Semenza, G.L.et.al., Mol.Cell.Biol.10:930-938 (1990)) that are normally at 609 bp fragment upstreams from genomic dna.For example, the fragment of available 609 bpPCR amplification is screened phage, Ke Shi plasmid or yeast artificial chromosome library as probe.Required clone will have one 6 kb BamHI-HindIII fragment, and can be by its restriction map and the people EPO gene of measuring with currently known methods restriction map are on every side compared its identity of susceptible of proof.In addition, also can use the restriction map of 609 bp fragments, derive between HUMERPA equivalent 2 and 609 and the segmental enzyme in BamHI site, extend through upstream identify to produce as the people's gene group upstream of probe manufacturing EPO gene; Available gel electrophoresis separates this fragment from human gene group DNA's suitable digestion product, and is connected in bacterium or the yeast clone carrier.Correct clone will and contain one 6 kb BamHI-HindIII fragment with 609 bp, 5 ' EPO probe hybridization.Isolating 6 kb fragments are being inserted (the HindIII site is adjacent to HUMERPA nucleotide position 2 like this) among p5 ' EPO, p5 ' EPO-mMTF or the p5 ' EPO-mMTS with suitable direction.Can use currently known methods such as karyomit(e) walking technology or separate and separate additional upstream sequence with the yeast artificial chromosome of 609 bp, 5 ' EPO probe hybridization.

Above-mentioned cloning process will allow the sequence upstream of EPO to be able to external modification, with continue after elementary, secondary or permanence human fibroblasts decided the target transfection.These methods are addressed the simple insertion of mMT promotor, and the disappearance of the disappearance of negative regulatory region and negative regulatory region and with having the displacement that the active enhanser of extensive host cell carries out.

G. activate people EPO gene and separate elementary, the secondary and permanence human fibroblasts of target with sieve method

In order to hit target, use the Restriction Enzyme cutting plasmid of the insertion section of from the plasmid major key, dissociating.When cutting p5 ' EPO-mMTS, what HindIII and SalI digestion discharged 2.4 kb hits the target fragment, and this fragment comprises by 405 bp of the DNA of the regulatory region that will be used to make this construct target people EPO gene and 204 base pairs and is attached to 5 respectively ' and 1.8 kb mMT promotors of 3 ' side.With phenol extraction and with this DNA of ethanol sedimentation purifying or 2.4 kb single hit the target fragment and under condition described in the embodiment 1c transfection in elementary or secondary human fibroblasts.The cells transfected shop is applied in the 150mm of human fibroblasts's nutritional medium plate.After 48 hours with cell with 10,000 cells/cm ²Density shop be applied to (about 20,000 cells in every hole in the 24 hole plates; If hit the target incidence is per 10 ⁶But one (embodiment 1c) arranged in the individual clone cell, then need to detect 50 holes) to isolate single expression colony.Wherein the transfection DNA cell that hit the upstream, homology zone of people EPO gene will be expressed hEPO under the control of mMT promotor.After 10 days, use the EPO expression level in the supernatant liquor of the whole hole of immunity test medicine box available on the market (Amgen) detection.The use currently known methods is isolated to derive from and is showed the cell clone that has in the hEPO synthetic hole, generally by detection be assigned to the allos cell colony in each hole or the plate each several part, detect the each several part in these positive holes and repeat where necessary, by screening with 96 hole titer plate of 1 cell inoculation in every hole the last colony that hits that separates.Also can use the DNA that derives from whole dull and stereotyped lysate in conjunction with the mMT Auele Specific Primer that is positioned at the primer of HUMERPA nucleotide position 1 upstream by PCR with amplified fragments analysis.This primer is to the big or small dna fragmentation that should be able to increase based on this dna sequence dna accurately predicting.Positive dull and stereotyped and with the extent of dilution that lowers in succession bed board again with tryptic digestion, target cell repeats DNA preparation and PCR step in separating as needs.

Also can use the target scheme of hitting as herein described to activate permanence people cell (as HT 1080 cells (ATCC CCL 121), the strain of deriving (the ATCC CCL 2 of Hela cell and HeLa cell, 2.1 and 2.2), MCF-7 breast cancer cell (ATCC HBT 22), K-562 leukemia cell (ATCC CCL 232), KB cancer cells (ATCC CCL 17), 2780 AD ovarian cancer cells (Van der Blick, A.M.etal., Cancer Res.48:5927-5932 (1988)), Raji cell (ATCC CCL 86), Jurkat cell (ATCC TIB 152), Namalwa cell (ATCC CRL 1432), HL-60 cell (ATCC CCL 240), Daudi cell (ATCCCCL 213), RPMI 8226 cells (ATCC CCL 155), U-937 cell (ATCC CRL1593), Bowes melanoma cells (ATCC CRL 9607), MI-38VA13 subbreed 2R4 cell (ATCC CLL 75.1), MOLT-4 cell (ATCC CRL 1582) and various different hybridoma) in hGH express the purpose that is used for the hGH that common medicine transmits with production.

H. activate people EPO gene and use the positive or bonded male/female selective system is separated elementary, the secondary and permanence human fibroblasts hit

Making up p5 ' EPO-mMTF, p5 ' EPO-mMTS and their strategy that has the additional segmental derivative of upstream 6 kbBamHI-HindIII can then carry out at this additional step of adjacent mMT promotor place insertion neo gene.In addition, can in the pBluescriptIISK/+ polylinker, insert a negative selection marker such as gpt (from pMSG (Pharmacia) or other appropriate sources) by HUMERPA sequence adjacent.In the previous case, separate with DNA that the Southern hybridization analysis makes from the colony gleanings and screen G418 through pcr amplification or Restriction Enzyme ^rColony is with the evaluation colony that hits.Under latter event, with G418 ^rThe integration (Besna-rd, C.et al., Mol.Cell.Biol.7:4139-4141 (1987)) of selecting the gpt gene in containing the xanthic substratum of 6-sulfydryl with contrast is applied in the colony shop.In addition, the HSV-TK gene can be placed on and insert the opposite sides of section, select neo and stop gpt and TK by culturing cell in the human fibroblasts's nutritional medium that contains 400 μ g/ml G418,100 μ M 6-sulfydryl xanthine and 25 μ g/ml gancyclovir with permission as gpt.Dual negative selection will provide almost the consequence that truly hits and select utterly, and the Southern engram analysis will provide last confirmation.

For production is suitable for the hEPO that conventional medicine transmits, also can use the target scheme of hitting as herein described to activate at permanence people cell (as HT 1080 cells (ATCC CCL 121), the strain of deriving (the ATCC CCL 2 of Hela cell and HeLa cell, 2.1 and 2.2), MCF-7 breast cancer cell (ATCC HBT 22), K-562 leukemia cell (ATCC CCL 232), KB cancer cells (ATCC CCL 17), 2780 AD ovarian cancer cells (Van der Blick, A.M.et al., Cancer Res.48:5927-5932 (1988)), Raji cell (ATCC CCL 86), Jurkat cell (ATCC TIB 152), Namalwa cell (ATCC CRL 1432), HL-60 cell (ATCC CCL 240), Daudi cell (ATCC CCL 213), RPMI 8226 cells (ATCC CCL 155), U-937 cell (ATCC CRL 1593), Bowes melanoma cells (ATCC CRL 9607), WI-38VA13 subbreed 2R4 cell (ATCC CLL 75.1), MOLT-4 cell (ATCC CRL 1582) and various different hybridoma) in hGH express.

I. be used for being placed on human growth hormone elementary, secondary or the structure that hits the target plasmid of permanence human fibroblasts under the control of mouse metal sulfur hydrochloride promotor

The following example is used to illustrate one embodiment of the invention, and wherein the normal sequence upstream of human growth hormone is changed, to allow expressing human tethelin in elementary, secondary or permanence human fibroblasts.

The fragment of cloned DNA that use is derived from 5 of human growth hormone N gene ' end produces and to be used to hit EPO generegulation district, similar to person described in the embodiment 1f target molecule that hits.The PCR primer that use designs based on analyzing the HUMGHCSA sequence in this zone is crossed over about 1.8 kb fragments of HUMGHCSA (Genbank Entry) nuclear former times acid position 3787-5432 (position in the segmental EcoRI site of size has been determined in two generations easily, and said fragment can be used for the clone or the property identified digestion comprises this segmental subclone) with amplification.This zone extends to the upstream position of about 1.4 kb of rotaring intertranslating start site 5 ' end from the middle part of hGH gene N introne 1.With EcoRI and BamHI digestion pUC12, handle producing blunt end with KLenow, and under the diluent condition recirculation, lost the plasmid in EcoRI and BamHI site.This plasmid is called as pUC12XEB.The HindIII joint is connected on the hGH fragment of amplification and, is connected on the pUC12XEB of HindIII digestion with HindIII digestion gained fragment.With EcoRI and BamHI digestion gained plasmid pUC12XEB-5 ' hGH, to remove the 0.5 kb fragment that is positioned at the upstream that is right after the hGH transcription initiation site.The DNA of digestion is connected on the 1.8 kb EcoRI-BglI fragments from the mMT-I gene [do not contain the mMT encoding sequence; Hamer, D.H.and Walling, M., J.Mol.Appl.Gen.1:273-288 (1982); Also can use the mMT sequence that derives from Genbank according to analysis is the PCR primer that MUSMTI, MUSMTIP, MUSMTIPRM design, and presses currently known methods and separate this fragment from mouse gene group DNA].This plasmid p5 ' hGH-mMT has the mMT promotor that is connected near on the hGH sequence both sides, upstream.

Above-mentioned clone's strategy can make the sequence upstream of hGH be modified external, so as continue after decide elementary, the secondary or permanence human fibroblasts of target transfection.This strategy has been described the simple insertion of mMT promotor.Also can give and see some other strategy, for example will have the upstream that the specific enhanser of extensive host cell is inserted into the mMT sequence of having inserted.

J. elementary, the secondary and permanence human fibroblasts who activates people hGH gene and hit with the screening method separation

In order to hit target, use the restriction enzyme cutting plasmid of the insertion section of from the plasmid main chain, dissociating.When cutting p5 ' hGH-mMT, HindIII digestion discharges the target fragment of 2.9 kb, this fragment by by the DNA side joint of regulatory region that makes this construct hit the hGH gene 5 ' and 3 ' side on 1.8 kb mMT promotors form.Hit the target fragment with phenol extraction and this DNA of ethanol precipitation purifying or 2.9 independent kb, and under the condition described in the embodiment 11 transfection in elementary or secondary human fibroblasts.Transfected cell shop is applied in containing the 150mm plate of human fibroblasts's nutritional medium.After 48 hours, with cell with 10,000 cells/cm ²Density shop be applied to (about 20,000 cells in every hole in the 24 hole wares; If hitting the ratio of target generation is per 10 ⁶Individual swelling has 1 (embodiment 1c) in the cell, then need detect about 50 holes for separating single expression colony).Wherein the transfection DNA cell that hit the homologous region upstream of hGH will be expressed hGH under mMT promotor control.After 10 days, the immunodetection medicine box (Nichols) that use can be buied from market detects the supernatant liquor in whole hole to determine the hGH expression level.Use the existing hGH synthetic clone of currently known methods separating table from the hole, normally be assigned to the each several part of the heterogeneous population of cell in single hole or the flat board by detection, detect the each several part in these positive holes, and repeat above-mentioned steps where necessary, finally separate the colony that hits with 96 hole titer plate of 1 cell inoculation in every hole by screening.Also can use the mMT Auele Specific Primer and be used in combination the primer in the downstream that is positioned at HUMGHCSA nucleotide position 5,432, derive from the DNA of whole dull and stereotyped lysate by the PCR method analysis of amplification of DNA fragments.This primer is to the big or small dna fragmentation that should be able to increase based on the accurately predicting of this dna sequence dna.Positive dull and stereotyped and with the extent of dilution that reduces successively bed board again with tryptic digestion, repetition DNA preparation in case of necessity and PCR step are with target cell in separating.

For producing the purpose of the hGH that conventional medicine transmits, as herein described hit the target scheme also can be used for activating hGH at the permanence cell (as HT 1080 cells (ATCC CCL 121), the strain of deriving (the ATCC CCL 2 of HeLa cell and HeLa cell, 2.1 and 2.2), MCF-7 mammary cancer (ATCC HBT 22), K-562 leukemia cell (ATCC CCL 232), KB cancer cells (ATCC CCL 17), 2780 AD ovarian cancer cells (Van dar Blick, A.M.et al., Cancer Res., 48:5927-5932 (1988)), Raji cell (ATCC CCL 86), Jurkat cell (ATCC TIB 152), Namalwa cell (ATCC CRL 1432), HL-60 cell (ATCC CCL 240), Daudi cell (ATCC CCL 213), RPMI 8226 cells (ATCC CCL 155), U-937 cell (ATCC CRL 1593), Bowes melanoma cells (ATCC CRL 9607), WI-38VA13 subbreed 2R4 cell (ATCC CLL 75.1), MOLT-4 cell (ATCC CRL 1582) and various different hybridoma) in expression.

K. activate people hGH gene and use the positive or bonded male/female selective system is separated elementary, the secondary and permanence human fibroblasts hit

The strategy that makes up p5 ' hGH-mMT can carry out the neo gene is inserted this additional step at adjacent mMT promotor place thereafter.In addition, can in the pUC12 polylinker, insert a negative selection marker such as gpt (deriving from pMSG (pharmacia) or other appropriate sources) in adjacent HUMGHCSA sequence place.In the previous case, separate with DNA that the Sout-hern hybridization analysis makes from the colony gleanings by pcr amplification or Restriction Enzyme and screen G418 ^rColony is with the evaluation colony that hits.Under latter event, with G418 ^rColony is placed on and contains the integration (Besmard, C.et al., Mol.Cell.Biol., 7:4139-4141 (1987)) of selecting the gpr gene in the xanthic substratum of sulfydryl with contrast.In addition, the HSV-TK gene can be placed on and insert the phase counter-example face of section, select neo and stop gpt and TK by culturing cell in the human fibroblasts's nutritional medium that contains 400 μ g/ml G418,100 μ m 6-sulfydryl xanthine and 25 μ g/ml gancyclovir with permission as gpt.Dual negative selection will provide the real consequence that hits almost and select utterly.The Southern hybridization analysis then is a confirmation property.

For production is suitable for the hGH that conventional medicine transmits, also can use the target scheme of hitting as herein described to activate permanence people cell (as HT 1080 cells (ATCC CCL 121), the strain of deriving (the ATCC CCL 2 of HeLa cell and HeLa cell, 2.1 and 2.2), MCF-7 mammary cancer (ATCCHBT 22), K-562 leukemia cell (ATCC CCL 232), KB cancer cells (ATCC CCL17), 2780 AD ovarian cancer cells (Van dar Blick, A.M.et al., CancerRes., 48:5927-5932 (1988)), Raji cell (ATCC CCL 86), Jurkat cell (ATCC TIB 152), Namalwa cell (ATCC CRL 1432), HL-60 cell (ATCC CCL 240), Daudi cell (ATCC CCL 213), RPMI 8226 cells (ATCC CCL 155), U-937 cell (ATCC CRL 1593), Bowes melanoma cells (ATCC CRL 9607), WI-38VA13 subbreed 2R4 cell (ATCC CLL 75.1), MOLT-4 cell (ATCC CRL 1582) and various different hybridoma) in hGH express.

Can modify described in embodiment 1f and the 1i and embodiment 1g, 1h, 1j and 1k in use hit the target construct, but but to make it to comprise the amplification property selection marker (as ada, dhfr or CAD) that is used to select the cell that the native gene that wherein is activated and amplification property selection marker all be amplified.Can use these cells of expressing the endogenous gene that maybe can express the coding therapeutic product to be used for the protein (as hGH and hEPO) of conventional medicine transmission or gene therapy with production.

1. use foreign DNA and alternative marker gene transfection primary and secondary inoblast with electroporation

With tryptic digestion and with nutritional medium exponential growth or inoblast of early stage stationary phase under the drip washing from the frosting.Take out the cell suspending liquid of a five equilibrium and count, carry out centrifugal remaining cell.The sucking-off supernatant liquor also is suspended in 5ml electroporation damping fluid (20mM HEPES pH 7.3,137mM NaCL, 5mM KCl, 0.7mM Na again with cell mass ₂HPO ₄, the 6mM dextrose) in.The recentrifuge cell, the sucking-off supernatant liquor is suspended in cell in the electroporation damping fluid that contains the 1mg/ml acetylated bovine serum albumin again.Last cell suspension contains has an appointment 3 * 10 ⁶Cell/ml.Again should carry out electroporation immediately after suspending.

In the aseptic Xiao Chi (Bio-Rad) that has the 0.4cm interelectrode distance, add super spirial plasmid DNA.The DNA final concentration is generally at least 120 μ g/ml.In Xiao Chi, add the 0.5ml cell suspension then and (contain 1.5 * 10 approximately ⁶Individual cell), and gently mixed cell suspension and dna solution.(Bio-Rad) carries out electroporation with the Gene-Pulser device.Electric capacity and voltage fix on 960 μ F and 250～300V respectively.Along with voltage increases, cell survivaling number reduces, but the per-cent that has the DNA of importing stably to mix the survivaling cell in its genome then significantly increases.Under these given parameters, should observe the burst length of about 14～20mSec.

The cell that is subjected to electroporation was at room temperature kept 5 minutes, use the aseptic whole pipet content of sucking-off Xiao Chi lightly then.Cell directly is added in the nutritional medium (the same, but be added with 15% calf serum) of the 10ml preheating in the 10cm plate and carries out incubation as mentioned above.Second day former substratum of sucking-off, and change the 10ml fresh culture into and continued incubation 16～24 hours.Second day again clone cell to determine cloning efficiency and to select G418 resistance colony.Use trypsin digestion and cell, counting and bed board; Be generally and determine that cloning efficiency is with 10 ³Inoblast is applied in cell/10cm plate shop; Select then with 1～2 * 10 for carrying out G418 ⁴Inoblast is applied in cell/10cm plate shop.

(Geneticin renders a service about 50% dithionate containing 300～400 μ g/ml G418; Gibco) select G418 resistance human fibroblasts in the inoblast nutritional medium (containing 15% calf serum).Do not having to determine cloning efficiency under the condition of G418.With bed board cell incubation 12～14 days, use the formalin colony between at this moment, with violet staining and counting (determining the cloning efficiency of bed board) or use clone's cylinder to separate (G418 flat board).Electroporation to rabbit fibroblast is identical with the human fibroblasts basically with selection, only is to use different selection conditions, selects the rabbit fibroblast to the G418 resistance in the substratum that contains 1gm/ml G418.

From people's foreskin of new cutting-out, be separated into fibrocyte.Density inoculation culture thing in the DMEM+10% calf serum with 50,000 cells/cm.When culture converges, also use the electroporation transfection through trypsin treatment results inoblast.Through estimating electroporation conditions through transfection with plasmid pcDNEO (Fig. 5).Use is near top condition (60 μ g plasmid pcDNEO, 250 volts of electroporation voltages, electric capacity fixes on 960 μ F) carry out the experiment of representational electroporation, per 588 the processed cells of result produce 1 G418 colony (all processed cells 0.17%), but or per 71 clone cells produce 1 G418 colony (1.4%).

When under top condition (60 μ g plasmid pcDNEO, 300 volts of electroporation voltages, electric capacity fix on 960 μ F) almost, advancing nine independent electroporations experiments, average per 1, observe 1 G418 colony (0.05%) in 899 processed cells, scope is 1/882 to 1/7,500 processed cell.But this is equivalent to per 38 clone cells 1 G418 colony (2.6%) is arranged on average.

Change the hGH express cell through making to pass at the low into for the primary human inoblast with plasmid pcDNEO and pXGH5 cotransfection.Generally be to carry out transfection with molar mixtures such as 60 μ g, two plasmids down in nearly top condition (electroporation voltage is 300 volts, and electric capacity fixes on 960 μ F).Experimental result is that per 14,705 processed cells produce 1 G418 colony.

The hGH expression data of isolating these and other cell is summarized as follows under identical transfection conditions.At last, all G418 ^r98% of colony can be expanded to produce a large amount of cultures.The G418 that analyzes ^rClone's number 154G418 ^rThe number 65 average hGH expression level 2.3 μ g hGH/10 of/hGH cloning by expression ⁶The maximum hGH expression level of cell/24hr 23.0 μ g hGH/10 ⁶Cell/24hr

Elementary or secondary human fibroblasts also can produce stable transfection body by electroporation transfection to use wherein neo and hGH gene to be present in DNA construct pXGH301 on the same plasmid molecule.Make up pXGH301 with two step methods.From pBR322, separate SalI-ClaI fragment (position 23～651 among the pBR 322) and be inserted among the pcDNEO of SalI-ClaI digestion the upstream, BamHI site in the SV40 early promoter zone of importing pcDNEO.Digest this plasmid pBNEO with BamHI, separation contains the 2.1kb fragment that is in the neo gene under the control of SV40 early promoter and inserts among the pXGH5 of BamHI digestion.Separate and have the plasmid that single 2.1 kb BamHI fragments are inserted, wherein neo and hGH transcribe with mutually the same direction.This plasmid is called as pXGH301.For example with 60 μ g pXGH301 electroporation 1.5 * 10 under 300 volts and 960 μ F conditions ⁶Individual cell.With per 1.5 * 10 ⁶The frequency of 652 G418 resistances of individual processing cell colony (handling 1 colony of cell for per 2299) is separated G418 resistance colony from transfected secondary inoblast.Have 59% to express hGH in these colonies approximately.

Embodiment 2 make up produce chimeric transcription units hit the target plasmid, merging in the chimeric transcription unit has human growth hormone and erythropoietin sequence

Following embodiment is used to illustrate two other embodiment of the present invention, wherein the normal regulating sequence upstream of people EPO gene is changed, but to allow under its former not transfected state, not expressing hEPO in the elementary or secondary inoblast cell strain with detection limit expression hEPO.In these embodiment schemes, the product that hits target is a chimeric transcription unit, and wherein first exon of human growth hormone gene is positioned at the upstream of hEPO exon 2～5.The product of transcribing, splicing and translate is the protein that amino acid/11～4 of wherein hEPO signal peptide are replaced by the amino-acid residue 1～3 of hGH.With regard to the relative position of the external adjusting sequence that is inserted into for producing the required specificity connecting method of final, processed transcript, these two embodiments are different.

Design plasmid pXEPO-10 hits the exons 1 that target is used the exons 1 displacement hEPO of hGH to carry out gene by the endogenous hEPO gene to human chromosome 7.Make up plasmid pXEPO-10 as follows.At first, the 6 kb HindIII-BamHI fragments (referring to embodiment 1f) that will be positioned at the hEPO upstream of coding region insert the pBluescri-ptIISK+ of HindIII-BamHI digestion (Stratagene, LaJolla, CA) in and make up intermediate plasmid PT163.Digest the product of this ligation and be connected to XhoI and HindIII from pMClneoPolyA[Thomas, K.R.and Capecchi, M.R.Cell 51:503-512 (1987), can derive from Stategene, LaJolla, CA] 1.1 kb HindIII-XhoI fragments on, to produce pT163.Utilize oligonucleotide 13.1～13.4 to merge fragment to produce one in polymerase chain reaction, wherein mouse metal sulfur hydrochloride I (mMT-I) promotor-hGH exons 1 sequence is fused on the hEPO introne 1 sequence extraly.At first, with oligonucleotide 13.1 and 13.3 with the mMT-I promotor-hGH exons 1 fragment of about 0.73 kb that increase from pXGH5 (Fig. 5).Then, about 0.57 kb fragment of mainly forming with oligonucleotide 13.2 and 13.4 amplifications by hEPO introne 1 among the human gene group DNA.At last, mixing two fragments that increased also further increases to produce last fusion fragment (merging fragment 3) with oligonucleotide 13.1 and 13.4, this merges the 5 ' side SalI site side joint of fragment in the mMT-I part, and at 3 ' side XhoI site of hEPO introne 1 sequence side joint.Merge fragment 3 and be connected to the pT163 that XhoI digests with XhoI and SalI digestion.This is connected mixture is transformed in the intestinal bacteria, and identify that containing merge fragment 3 single inserts the clone of section and name and be pXEPO-10, in described fusion fragment 3 in 3 of hEPO introne 1 sequence ' stress newly to have produced XhOI site.13.1 5′TTTTGTCGAC?GGTACCTTGG?TTTTTAAAAC?C

SalI KpnI

(SEQ?ID?NO?5)13.2 5′CCTAGCGGCA?ATGGCTACAG?GTGAGTACTC?GCGGGCTGGG?CG

(SEQ?ID?NO?6)13.3 5′CGCCCAGCCC?GCGAGTACTC?ACCTGTAGCC?ATTGCCGCTA?GG

(SEQ?ID?NO?7)13.4 5′TTTTCTCGAG?CTAGAACAGA?TAGCCAGGCT?G

XhoI

(SEQ?ID?NO?8)

The territory, non-black-body block of oligomer 13.1 is identical with the mMT-I promotor, has the natural KpnI site as its 5 ' border.The boldface font representative is to be the SalI site afterbody in SalI site with 5 ' boundary transition.Oligomer 13.2 and 13.3 boldface type Regional Representative hGH sequence, but not the boldface type zone is the introne 1 sequence from the hEPO gene.The territory, non-black-body block of oligomer 13.4 is identical with last 25 bases of hEPO introne 1.The boldface type zone comprises that 3 ' boundary transition with amplified fragments is the XhoI site afterbody in XhoI site.

Design plasmid pXEPO-10 places endogenous hEPO seat on the human chromosome 7 to hit target by gene with the mMT-I promotor of the hGH upstream of hEPO structure gene and promoter region and exons 1.Make up plasmid pXEPO-11 as follows.Utilize oligonucleotide 13.1 and 13.5～13.7 to merge fragment to produce one in polymerase chain reaction, mouse metal sulfur hydrochloride I (mMT-I) promotor-hGH exons 1 sequence is fused on the hEPO sequence with respect to hEPO coding region from-1 to-630 extraly in this fragment.At first, increase from about 0.75 kb mMT-I promotor-hGH exons 1 fragment of pXGH5 (Fig. 5) with oligonucleotide 13.1 and 13.6.Then, with oligonucleotide 13.5 and 13.7 amplifications from the human gene group DNA, with respect to about 0.65 kb fragment of mainly forming of hEPO coding region by from-1 to-620 hEPO sequence.Oligonucleotide 13.5 and 13.6 all contains the 10bp joint sequence that is positioned at hGH introne 1-hEPO promoter region, and it is equivalent to natural hEPO introne 1 splice donor site.At last, mix two fragments that increased and further use oligonucleotide 13.1 and 13.7 amplifications, to produce last fusion fragment (merging fragment 6), 5 ' side Sa1I site of this fragment side joint mMT-I part, and 3 ' side XhoI site of side joint hEPO promoter region.Merge fragment 6 and be connected on the PT163 of XhOI digestion with XhoI and SalI digestion.To connect mixture and be transformed in the intestinal bacteria, and identify and contain the single clone who inserts section of merging fragment 6, and name and be pXEPO-11, in the said fusion fragment 6, on 3 ' side of hEPO promoter sequence, produce the XhoI site again.13.5 5′GACAGCTCAC?CTAGCGGCAA?TGGCTACAGG?TGAGTACTC

AAGCTTCTGG?GCTTCCAGAC?CCAG (SEQ?ID?NO?9)

HindIII13.6 5′CTGGGTCTGG?AAGCCCAGAA?GCTTGAGTAC?TCACCTGTAG

HindIII

CCATTGCCGC?TAGGTGAGCT?GTC (SEQ?ID?NO?10)13.7 5′TTTTCTCGAG?CTCCGCGCCT?GGCCGGGGTC?CCTC

XhoI

(SEQ?ID?NO?11)

The hGH sequence is represented in oligomer 13.5 and 13.6 black matrix block.The zone of italics is equivalent to preceding 10 base pairs of hEPO intron.The oligomer rest part is equivalent to the hEPO sequence of from-620 to-597 (being equivalent to the hEPO coding region).The territory, non-black-body block of oligomer 13.7 with respect to the base-1 of hEPO coding region to-24 identical.The boldface type zone comprises that the segmental 3 ' boundary transition that will increase is the XhoI site afterbody in XhoI site.

Available BamHI and XhoI digested plasmid pXEPO-10 hit target to discharge the 7.3kb fragment (wherein said syzygy is in its both sides side joint hEPO sequence) that contains the mMT-I/hGH syzygy and this plasmid is used for gene.This fragment (hitting target fragment 1) does not contain the hEPO encoding sequence, only is positioned at-620 peace treaty-6620 sequences of the upstream of hEPO coding region and hEPO introne 1 sequence, to instruct people EPO seat is hit target.Use the simulated condition described in the embodiment 1c, will hit 1 transfection of target fragment in the primary and secondary human skin fibroblast.G418 resistance colony is inoculated in each holes of 96 hole flat boards and with ELISA method (R﹠amp; D system, Minnea-polis, MN) screening is to express EPO.Wherein the transfection DNA random integration can not produce EPO to the cell in the people's gene group.Wherein transfection DNA contains a mosaic gene with the cell of endogenous hEPO introne 1 and hEPO upstream sequence generation homologous recombination, and mMT-I promotor and the sequence of not transcribed and hGH 5 ' do not translate sequence and the hGH exons 1 has replaced normal hEPO promotor and hEPO exons 1 (see figure 1) in this gene.Hit non-hEPO sequence in the target fragment 1 and be connected to the hEPO sequence in hEPO introne 1 downstream.To activate EPO gene in the inoblast of not expressing hEPO under the normal circumstances with the normal hEPO regulatory region of mMT-1 promoter replacement.Replace the hEPO exons 1 with the hGH exons 1 and then produce the protein that preceding 4 amino acid of hEPO signal peptide are wherein replaced by the amino acid/11 of hGH～3, form a functional chimeric signal peptide, this signal peptide is removed and makes it to secrete from express cell when translating post-treatment from mature protein.

Hit target by making plasmid pXEPO-11 be used for gene, to discharge the 7.4kb fragment that contains the mMT-I/hGH syzygy of side joint hEPO sequence on its both sides with BamHI and XhoI digestion.This fragment (hitting target fragment 2) does not contain the hEPO encoding sequence, and only has the sequence of between upstream-1 peace treaty-6620 that is positioned at the hEPO coding region, to instruct people EPO seat is hit target.Use and to hit 2 transfections of target fragment in the first utmost point or secondary human skin fibroblast to condition similar described in the embodiment 19.G418 resistance colony is collected in the single hole of 96 hole flat boards, and with ELISA detection method (R﹠amp; D system, Minneapolis, MN) screening has the colony that EPO expresses.Wherein the transfection DNA random integration can not produce EPO to the cell in the people's gene group.Wherein transfection DNA contains a mosaic gene with the cell of endogenous hEPO promotor and upstream sequence generation homologous recombination, in this gene the mMT-1 promotor and not transcription sequence, hGH5 ' do not translate sequence and hGH exons 1, and be inserted in the HindIII site (referring to Fig. 2) that is positioned at respect to the position-620 of hEPO coding region by 10 base pair joints of preceding 10 based compositions of hEPO introne 1.The mMT-I promotor located upstream of normal static hEPO promotor will in elementary or secondary skin flbroblast, instruct signal read sign indicating number (5 ' to 3 ') do not translate metallothioneins and hGH sequence, hGH exons 1, with the hEPO introne 1 before 10 10 DNA bases that base pair is identical, and the synthesizing of normal hEPO promotor and hEPO exons 1 (be equivalent to hEPO encoding sequence-620 to+13).As splice donor site the hGH exons 1 is fused to from 10 base pair joint sequences of hEPO introne 1 and is positioned at the acceptor splicing site site, next downstream that is right after hEPO exon 2 upstream.Therefore, the processing of gained transcript is fallen hEPO promotor, exons 1 and introne 1 sequence with splicing.To produce the protein that a kind of preceding 4 amino acid of wherein hEPO signal peptide replace with hGH amino acid/11～3 with hGH exons 1 displacement hEPO exons 1, one of this replacement formation be removed from mature protein by translating post-treatment and from express cell by the functional chimeric signal peptide of excretory.

Utilize currently known methods can make up a series of constructs relevant with pXEPO-10 and pXEPO-11.In these constructs, the relative position of mMT-1 promotor and hGH sequence, and the position of mMT-1/hGH sequence insertion hEPO upstream sequence changes, thereby produce selectable, be convenient to the mosaic type transcription unit that gene hits target, cause merging the more effective expression of transcript, perhaps other required characteristic of tool.This class construct will produce similar result, so the hGH-hEPO fusion gene is placed in by gene and hits under the regulation and control of the exogenous promoter that target to normal hEPO gene location produced.For example, 6 kb HindIII-BanHI fragments (referring to embodiment 1f) of hEPO upstream region of gene have and manyly can merge the Restriction Enzyme recognition sequence in segmental site as inserting neo gene and mMT-1 promotor/hGH.The BglII site that is positioned at about 1.3 kb in upstream, HindIII site is a said site, it is unique in this zone, and can be used for inserting one or more selected markers and will be used to activate the regulatory region of the gene that hEPO expresses from another kind in elementary, secondary or permanence people cell.

At first, insert the pBluescriptIISK+ that digested by HindIII-BamHI (St-ratagene, LaJolla, interstitial granules pT164 CA) and in making up by the 6 kb HindIII-BamHI fragments (embodiment 1f) that will be positioned at the hEPO upstream of coding region.With BamHI and XhoI digested plasmid pMClneoPloyA[Thomas, K.R.and Capecchi, M.R.Call 51:503-512 (1987); Can derive from Stratagene, LaJolla, CA], handle to make it to become blunt end with the KLenow fragment of e. coli dna polymerase, and the 1.1kb fragment of purifying gained.PT164 digests with BglI, and becomes blunt end through the processing of the Klenow of E.coli archaeal dna polymerase fragment.Connect above-mentioned two blunt end fragments, and be transformed among the competent E.coli.Separate and have the segmental single clone who inserts section of 1.1 kb neo, and analyze with the Restriction Enzyme analytical method, be the clone who is arranged in apart from unique HindIII site 1.3 kb that plasmid pT164 exists to determine that those wherein merge the BglI site that is rebuild by tack Xhol and BgIII site.So far can be at the plasmid pT165 of the unique BglII site cracking gained that is positioned at neo transcription unit 5 ' flank.

Oligonucleotide 13.8 and 13.9 be used in the polymerase chain reaction with produce one wherein have mouse metal sulfur hydrochloride I (mMT-I) promotor-hGH exons 1 sequence in addition also with the fragment of the 10 bp fragments fusion that contains splicing-donor site.Although have a large amount of splicing-donor sites or total splicing-donor site to be used, selected splicing-donor site is equivalent to natural hEPO introne 1 splicing-donor site.Oligonucleotide (13.8 and 13.9) the about 0.73 kb mMT-1 promotor-hGH exons 1 fragment (Fig. 5) that is used to increase from pXGH5.Digest the fragment (fragment 7) that is amplified with BglII, and be connected with the pT165 that digests through BglII.Connecting mixture is transformed among the E.coli, evaluation contains the single clone who inserts section of fragment 7, wherein, KpnI site in the mMT-1 promotor is adjacent to 5 of neo gene ' end, and the mMT-1 promotor is wanted so that transcribe direction location towards unique HindIII site, and this clone is named as pXEPO-12.13.8 5′AAAAAGATCT?GGTACCTTGG?TTTTTAAAAC?CAGCCTGGAG

BglII KpnI

(SEQ?ID?NO?12)

The non-black-body district of oligomer 13.8 is equal to the mMT-1 promotor, is its 5 ' border with natural KpnI site.The black matrix type represents with 5 ' boundary transition to be the BglII site tail in BglII site.13.9 5′TTTTAGATCT?GAGTACTCAC?CTGTAGCCAT?TGCCGCTAGG

BglII

(SEQ?ID?NO?13)

The black matrix district of oligomer 13.9 represents the hGH sequence.The italic block is equivalent to preceding 10 base pairs of hEPO introne 1.The BglII site that adds line is to make up the usefulness of plasmid.

Plasmid pXEPO-12 is used to gene, and to hit target be by with BamHI and HindIII digestion, contains by the 7.9 kb fragments of hEPO sequence side joint in the neo of both sides gene and mMT-1/hGH syzygy to discharge.This fragment (hitting target fragment 3) does not contain the hEPO encoding sequence, and the sequence that only contains between hEPO upstream of coding region about-620 to about-6620 is hit target to instruct to people EPO locus upstream.Use is similar to the condition described in embodiment 1b and the 1c will hit 3 transfections of target fragment in the human skin fibroblast of elementary, secondary or permanence, collect G418 resistance colony in each hole of 96 hole flat boards, and with ELISA assay method (R﹠amp; DSystems, Min-neapolis MN) screening EPO expresses.Have the transfection DNA random integration and can not produce hEPO to the cell of people's gene group, the DNA of transfection and endogenous hEPO promotor and upstream sequence carry out the cell of homology group, contain a mosaic type gene, in this mosaic type gene, mMT-1 promotor and non-transcribed sequence, hGH 5 ' do not translate sequence, hGH exons 1 and are inserted in the BglII site that is positioned at about-1920 positions relevant with the hEPO coding region with 10 bp joints by preceding 10 based compositions of hEPO introne 1.Synthetic following message content in elementary, secondary or permanence human fibroblasts (or other people's cell) will be instructed in the location of the mMT-1 promotor of normal immobilized hEPO promotor upstream: the metallothioneins that does not translate (5 ' → 3 ') and hGH sequence, hGH exons 1, DNA 10 bases identical with preceding 10 base pairs of hEPO introne 1 and hEPO upstream and hEPO exons 1 (be correlated with the EPO encoding sequence about-1920～+ 13).10bp joint sequence from the hEPO introne 1 plays splicing-donor site effect, so that the hGH exons 1 merges with the downstream splicing-acceptor site that is in close proximity to hEPO exon 2 upstream.Therefore, the processing of gained transcript is fallen hEPO upstream sequence, promoter region, exons 1 and introne 1 sequence with splicing.When use pXEPO-10 ,-11 and-12 the time, the improvement of transcribing the post-treatment process of information, can be undertaken by utilizing the vitro mutagenesis method, to remove the acceptor splicing site site in the hEPO upstream sequence between mMT-I promotor and hEPO exons 1, this can reduce the degree that produces the necessary productivity splicing of information needed phenomenon.Substitute the hEPO exons 1 with the hGH exons 1 and produce protein, wherein the amino acid/11 of hGH～3 have replaced preceding 4 amino acid of hEPO signal peptide, the mosaic type signal peptide of systematic function, and this signal peptide is removed from sophisticated protein owing to the course of processing after translating, and by secreting in the express cell.

The amplification of the hit modification and the target gene of embodiment 3 upstream sequences

Contain because of the gene amplification phenomenon and can give its marker gene if hit the target plasmid, then just can induce wherein people's cell by preceding method activated hEPO gene with amplification neo/mMT-1/EPO transcription unit to high-level cytotoxic factor resistance.The marker gene that can screen such as Tetrahydrofolate dehydrogenase (dhfr, selective agent are methotrexates), multi-functional CAD gene [encoding carbamoyl phosphate synthetase, aspartate transcarbamylase base enzyme and dihydro whey enzyme (orotase); Selective agent is N-(phosphono-ethanoyl)-L-aspartic acid (PALA)], glutamine synthetase [selective agent is first sulfonyl ammonia sulphoximine (MSX)] and adenosine deaminase (ada, selective agent is an adenosine) the existing document record (Wright in other gene that in the human cell line of permanence, can increase, J.A.et al, Proc Natl.Acad.Sci.USA 87:1791-1795 (1990); Cockett, M.I.et al., Bio/Technology 8:662-667 (1990)).In these researchs, record gene amplification betides among many permanence human cell lines.Under suitable screening conditions, HT 1080, the Hela in other cell, MCF-7 breast cancer cell, K-562 leukemia cell, KB cancer cells or 2780 AD ovarian cancer cells show amplified reaction.

Plasmid pXEPO-10 and pXEPO-11 can be modified by the dhfr gene that inserts a normal or sudden change in unique HindIII site of these plasmids.With suitable DNA transfection HT 1080 cells, screen its G418 resistance (giving) and identify that hEPO gene is wherein carried out that gene hits target and behind the activated cell by neo, dhfr and mMT-1 sequence to the tram of hEPO upstream region of gene by the neo gene, these cells can be placed in the methotrexate (MTX) and screen step by step with the amplification of screening dhfr and the common amplification (Kaufman, R.J.Technique 2:221-236 (1990)) of connected neo, mMT-1 and hEPO sequence.In the substep screening scheme that is adopted, cell at first contacts with lower concentration MTX (0.01～0.08 μ M), and is cumulative until 250 μ M or higher MTX Continuous Contact with concentration subsequently.Though it also is effectively that the low relatively increment of MTX concentration changes, it will be favourable that the linear incremental step of 0.04～0.08 μ M MTX and the continuous duple of MTX concentration are increased in the transfectional cell series that screens amplification.Amplified reaction is monitored in increase by the dhfr gene copy number, and expresses by the outer hEPO of detection bodies and to confirm.By means of above-mentioned strategy, by obtaining hEPO expression in fact over one's competence to the modification that hits that is positioned at the sequence outside the hEPO coding region fully.

In order to hit the cell that target non-coding sequence and amplified reaction subsequently obtain overexpression hGH gene by gene, be similar to (embodiment 1f, 1h, 1i, 1k, 2 and 7) as herein described and also can be made it to comprise the dhfr gene in people's cell by further the modification with the construct that activation hGH expresses.

Embodiment 4 hits target and activates people EPO locus in the permanence human fibroblast cell line

The hypothesis that target construct pXEPO-13 can be activated with test endogenous hEPO gene is hit in preparation in the human fibroblasts.At first, make up plasmid pT22.1, it contains 63 bp genome hEPO sequences of first codon upstream of the hEPO gene that merges with mouse metal sulfur hydrochloride-1 promotor (mMT-I).Oligonucleotide 22.1 to 22.4 is used among the PCR to merge mMT-I and hEPO sequence.The characteristic of these primers is as follows: 22.1 is oligonucleotide of one 21 base, the 28 bp fragment homologies that begin with the mMT-I promotor of upstream, mMT-IKpnI site; 22.2 and 22.3 are complementary primers of 58 Nucleotide, they determine the syzygy of hEPO and mMT-I sequence, make 28 bp of 35 bases that hEPO sequence that this syzygy contains first codon upstream of hEPO gene begins, with the mMT-I sequence of the base 29 that starts from oligonucleotide 22.2, it comprises the natural B glII site of mMT-I and extends to 30 bases of mMT-I sequence; 22.4 be long 21 Nucleotide, and the 725 bp homologies that begin with the hEPO sequence in the downstream of first codon of hEPO gene.These primers are used to increase and contain 1.4 kb dna fragmentations of above-mentioned mMT-I and hEPO sequence syzygy.With the fragment (this PCR fragment contains two KpnI sites: the single natural KpnI site of mMT-T promoter region and the single natural KpnI site in the hEPO sequence) of KpnI digestion gained, and carry out purifying.Plasmid pXEPOI also with KpnI digestion, discharges one 1.4 kb fragment and one 6.4 kb fragment.Purifying 6.4 kb fragments, and merge fragment with 1.4 kb KpnI PCR and be connected.The construct of gained is called pT22.1.The structure of second middle interstitial granules pT22.2 is pBSIISK+ (Stratagene, LaJolla, CA) connection by about 6 kb HindIII-BamHI fragments (referring to embodiment 1f) that will be positioned at hEPO structure gene upstream and BamHI and HindIII digestion.Make up the 3rd middle interstitial granules pT22.3 and be at first from the pMCINEpolyA that contains neomycin phosphotransferase gene (Stratagene, LaJolla, CA) in the XhoI/BamHI fragment of one 1.1 kb of excision.Using the Klenow fragment (New England Biolabs) of dna polymerase i that above-mentioned fragment is made tack does not then hold.Again this fragment is connected to the HincII site (also making tack with dna polymerase i similarly) of pBSIISK+ and obtains pT22.3.The preparation of the 4th middle interstitial granules pT22.4 is by the 1.1 kb XhoI/HindIII fragments that contain neo gene of purifying from pT22.3, and this fragment is linked to each other with pT22.2 through XhoI and HindIII digestion.Therefore pT22.4 contains the neo gene adjacent with the HindIII side of hEPO fragment upstream BamHI-HindIII.At last, obtain pXEPO-13 by at first from pT22.1, excising 2.0 kb EcoRI/AccI fragments.5 ' border of mMT-I promotor has been determined in this segmental EcoRI site, and this segmental AccI site then is arranged in hEPO exon 5.Therefore, this AccI/EcoRI fragment contains almost complete hEPO ceneme except the part and natural polyadenylation site that only lack exon 5.The above-mentioned 2.0 kb EcoRT/AccI fragments of purifying are handled with the Klennow fragment of dna polymerase i and to be made blunt end, are connected with pT22.4 through XhoI digestion and blunt endization then.

With pXEPO-13 transfection HT 1080 cells through PvuI-BamHI digestion.The pXEPO-13 of digestion produces three fragments in this way: the 1 kb carrier segments that comprises the amp Gene Partial; 1.7 kb fragments of the carrier sequence that stays and the about 9 kb fragments that contain hEPO, neo and mMT-I sequence.Described about 9 kb BamHI/PvuI fragments are begun to contain following sequence successively by the BamHI site: about 5.2 kb of hEPO genome sequence upstream, 1.1 kb noe transcription units, 0.7 kb mMT-I promotor and contain 2.0 kb fragments of the hEPO encoding sequence of brachymemma in exon 5.45 μ g are used to 1.2 * 10 through the pXEPO-13 of aforesaid method digestion ⁷In the electroporation of cell (condition of electroporation is described among the embodiment 1b).This electroporation process repeats 8 times altogether, forms altogether 9.6 * 10 ⁷The electroporation of cell.Cell mixes to produce 1 * 10 with substratum ⁶The cell density of cell/ml, the sample aliquot of getting 1 ml are dispensed to altogether in 96 the 150 mm tissue culturing plates (Falcon), and each contains the MIN DMEM/15% bovine serum of 35 ml.Second day,, and change old substratum with the fresh culture that contains 0.8 mg/ml G418 (Gibco) with the substratum sucking-off.Cultivate after 10 days, with elisa assay (R﹠amp; D Systems) hEPO in the substratum of each culture plate of sampling analysis.There are 6 in 96 flat boards and contain at least 10 mU/ml hEPO.Select in these culture plates, promptly No. 18 plate expressed colony with purifying hEPO.Each 150mm culture plate of 96 plates contains 600 the G418 resistance colonies of having an appointment (on 96 all flat boards, estimating always to have 57,600 G418 resistance colonies).About 600 colony trypsin treatment on No. 18 culture plates, and with the density of 50 cell/ml again bed board go in 364 orifice plates (Steril-in).After cultivating a week, in each macropore of 364 orifice plates, there are 10 colonies can individually see (these culture plates by form by 16 apertures in each macropore of 24 macropores) approximately.The expression that screen hEPO in each hole this moment.Include the substratum that has 20mU/ml hEPO at least in two macropores.In the A2 hole, find to contain 15 colonies that are distributed in 16 apertures.Content in each these aperture is all used trypsin treatment, and transfer in 16 independent holes of 96 culture plates.After cultivating 7 days, the hEPO elisa assay is carried out in sampling from these every hole substratum.Have only a hole (No. 10 holes) to contain HEPO.This cell strain is named as HT165-18A2-10, makes it breed in cultivation that quantitative hEPO analyzes to carry out, RNA separates and separates with DNA.The detection by quantitative result of hEPO productive rate is 2,500,000,000 units/10 ⁶Cell/24 hour.

Be used to survey isolated RNA from the HT165-18A2-10 cell by the AccI site to 3 in the hEPO exon 5 ' non-0.2kb dna probe of translating the BglII site in district.Do not contained these ACCI/BglII sequences in the AccI of exon 5 site by the target construct pXEPO-13 that hits of brachymemma, therefore, this construct has judgement for the target that hits at the hEPO locus.Only the cell strain of recombinating with homology mode and natural hEPO sequence can produce the hEPO mRNA that contains with the sequence of AccI/BglII sequence homology.Found HT165-18A2-10 be expressed in the Northern trace with ³²The mRNA of the pre-sizing of the AccI/BglII hEPO probe hybridization of P mark.Restriction Enzyme and Southern engram analysis confirm that neo gene and mMT-I promotor are hit on the seat of one of target to two hEPO allelotrope in the HT165-18A2-10 cell.

The above results shows, can a regulatory region be hit on target to a gene that remains static in the human fibroblasts usually with homologous recombination, thereby cause the functional activation of this gene.22.1 5′CACCTAAAAT?GATCTCTCTG?G (SEQ?ID?NO?14)22.2 5′CGCGCCGGGT?GACCACACCG?GGGGCCCTAG?ATCTGGTGAA

GCTGGAGCTA?CGGAGTAA (SEQID?NO?15)22.3 5′TTACTCCGTA?GCTCCAGCTT?CACCAGATCT?AGGGCCCCCG

GTGTGGTCAC?CCGGCGCG(SEQ?ID?NO?16)22.4 5′GTCTCACCGT?GATATTCTCG?G?(SEQID?NO?17)

The gene of embodiment 5 preparation intronlesses

Gene hits target and also can be used to prepare the gene that does not contain intron after processing, carries out genetic expression and external protein production in yeast or the bacterium to transfer to.For example, utilize following method to produce hGH by yeast.

Prepare two isolating target constructs that hit.Hit target construct 1 (TC1) and comprise the one section retroviral LTR sequence LTR of Moloney Murine leukosis virus (MoMLV) (for example from), one is used for the marker gene (as the neo gene from Tn5) of screening at people's cell, one is used for the marker gene (as yeast URA3 gene) of screening at yeast, energy in yeast, instruct genetic expression regulatory region (as the GAL4 promotor) and can be randomly one section sequence (leader sequence) that when with the hGH gene fusion, can make yeast cell secretion hGH.This carrier can comprise that also permission carries out the dna sequence dna of retrovirus packing in people's cell.Set up above-mentioned construct make above-mentioned sequence by hGH genome sequence side joint in both sides, when carrying out homologous recombination with genome hGH gene N sequence, this hGH genome sequence will be integrated exogenous array in hGH gene N codon 1 (corresponding to sophisticated, processed proteinic 1 amino acids) is right after the TCI of upstream.The order of the dna sequence dna in the integration is: the downstream sequence of hGH upstream and adjusting sequence, neo gene, LTR, URA3 gene, GAL4 promotor, yeast leader sequence, the hGH sequence that comprises maturation protein 1 amino acids and mature protein 1 amino acids.Hit target construct 2 (TC2) comprises to be enough to sequence (for example 2-micron circle (2-micron circle) or ARS sequence) yeast transcription termination sequence, the viral LTR that plasmid is duplicated and to be used for screening at people's cell in yeast marker gene (for example, bacterium gpt gene).Set up this construct and make the both sides of above-mentioned sequence by hGH genome sequence side joint, when carrying out homologous recombination with genomic hGH gene N sequence, this hGH genome sequence will be integrated exogenous array in the TC2 that is right after the downstream of hGH gene N terminator codon.The order of the dna sequence dna during integration is: the non-sequence of translating of hGH exon 5 sequences, yeast transcription termination sequence, yeast plasmid replication sequence, LTR, gpt gene and hGH3 '.

Linear fragment from TC1 and TC2 is hit the separately position of target to its hGH gene flank in succession.After a little cells being carried out superingection, instruct the LTR that transcribes to produce a RNA who has the LTR sequence at two ends by this district with complementary retrovirus.Splicing this RNA is removed normal hGH intron in the molecule that produces.The reverse transcription of processed transcript will cause the accumulation of the double-stranded DNA copy of processed hGH fusion gene.From by DNA isolation the dual cell that hits target, retroviral infection, and the enzyme that is used in disposable cracking transcription unit among the LTR digests.The material that is digested connects under the condition that can promote cyclisation, and is imported in the yeast cell, and the cell of gained screens the URA3 gene subsequently.Have only those cells of having taken in URA3 gene (being connected on the hGH gene of the sequence that imports by TC1 and TC2 and processing) to grow.These cells contain will express the proteic plasmid of hGH when semi-lactosi is induced, and be listed as from cell secretion hGH albumen by means of Fused yeast leading peptide, in case said leader peptide sequences is sophisticated in the secretion generation, have bioactive hGH to divide the period of the day from 11 p.m. to 1 a.m then cleavedly fall.

The simple replacement of expression in bacterial cell by in TC1 and TC2, carrying out, as use from the ampicillin resistance gene of pBR322 replace yeast URA3 gene, with tac promotor (deboer et al, Proc.Natl.Acad.Sci.80:21-25 (1983)) replace yeast GAL4 promotor, with bacterial leader sequences replace the yeast leader sequence, with the replication origin of pBR322 replace 2-micron circle or ARS sequence and with the bacterium Transcription Termination (as the trpA transcription terminator; Christic, G.E.et al., Proc.Natl.Acad.Sci.78:4180-4184 (1981)) sequence replaces the yeast transcription termination sequence to finish.Similarly, hit target sequence with hEPO and replace hGH to hit target sequence simply also expressing hEPO in yeast and bacterium, like this, yeast or bacterial leader sequences are positioned to be right after the upstream of hEPO codon 1 (1 amino acids in sophisticated preserved egg white matter mutually).

The embodiment 6 EPO gene that in the permanence human cell line, activates and increase

Unique HindIII site (referring to embodiment 4) that the dhfr ceneme is incorporated into pXEPO-13 will produce one can double selection and select the new hit target carrier of the cell that dhfr gene wherein is amplified.Single HindIII site among the pXEPO-13 defines being connected between the genome sequence of neo gene and the natural place upstream of people EPO gene.The dhfr gene is placed in provides one to have the neo that held by the dna sequence dna from natural hEPO gene location and the construct of dhfr gene on this site.The same with pXEPO-13, the derivative that inserts the dhfr gene is used to hit target hEPO locus through homologous recombination.This construct that is named as pREPO4 is shown among Fig. 6.This plasmid comprises people EPO gene extron 1～4 and part exon 5 and the HindIII-BamHI fragment that is positioned at the hEPO upstream of coding region.PSVe, pTK and pmMT-I be equivalent to from SV40 distinguish in early days, the promotor of simple sore exanthema virus (HSV) thymidine kinase (TK) gene and mouse metal sulfur hydrochloride-I gene.Its production process is as follows: the pXEPO-13 of purifying HindIII digestion makes it to become tack with the Klenow fragment of dna polymerase i.For obtaining the dhfr ceneme, with EcoRI and SalI digested plasmid construct pF8CIS9080 (Eaton et al., Biochemistry 25:8343-8347 (1986)).Contain 2 kb fragments of dhfr ceneme by purifying in the Digestive system, and handle with the Klenow fragment of dna polymerase i and to make it to become tack.Fragment with the above-mentioned dhfr of containing is connected with the tack HindIII site of pXEPO-13 then.The five equilibrium of getting this connection mixture partly is transformed into E.coli, and is plated on the penbritin selection flat board.After 37 ℃ of overnight incubation, observe, choose and cultivate single bacterium colony.Obtain the miniplasmids prepared product by these cultures, digest the DNA of gained then with BglI+HundIII and SfiI Restriction Enzyme, with the segmental direction of the dhfr that determines to be inserted into.The plasmid DNA that discovery derives from one of above-mentioned prepared product contains such dhfr fragment 2 kb insertion section.And the transcriptional orientation of finding dhfr ceneme in this plasmid is relative with the direction of the neo gene of adjacency.This is the construct that is named as pREPO4.

After activating endogenous hEPO gene through homologous recombination, plasmid pREPO4 is used to amplification hEPO locus in cell.The gene activation that produces with this construct makes and can select the DHFR of increase to express by utilizing medicine methotrexate (MTX).Generally speaking, the raising of DHFR expression realizes by DNA cloning increase copy number.Final result is that the hEPO gene that is activated is with dhfr sequence coamplification.The raising that the coamplification of activated EPO locus will cause EPO to express.

In HT 1080 cells, hit the target experiment with pREPO4.Separating the hEPO expression is HTREPO-52.EPO with Southern and the above-mentioned clone generation of Northern trace quantitative analysis.Find that this bacterial strain will be hit target by the dhfr/neo/mMT-I sequence of a single copy.The expression level that obtains under 0.8mg/ml G418 selection condition is about 1300mU/1 * 10 ⁶Cell/sky.Because target EPO locus contains a dhfr ceneme, might select the raising of DHFR expression with antifolic thing MTX.Therefore, this bacterial strain carries out the substep selection in 0.02,0.05,0.1,0.2 and 0.4 μ M MTX.Its initial selection result is listed in table 4 and Fig. 7.

Table 4 clone MTX (μ M) mU/1 * 10 ⁶Cell/24 hour 52C20-5-0 0 136852C20-5-0.01 0.01 174452C20-5-0.02 0.02 1164352C20-5-0.05 0.05 2444952-3-5-0.10 0.1 3701952-3-5-0.20 0.2 6786752-3-5-0.4B 0.4 99919

With the expression that the selection of the MTX concentration that increases has successfully improved hEPO among the clone HTREPO-52, observe for anti-0.4 μ M MTX that EPO output has increased by 70 times in this clone.Analyze in the MTX resistant cell line with the Southern trace and to confirm the amplification of hEPO locus, it reflects that activated hEPO locus has approximately increased by 10 times with respect to its parent (not hit target) its copy number of hEPO allelotrope.

Embodiment 7 produces the hEPO fusion gene by the CMV promotor of inserting genome hEPO upstream of coding region 1.8 kb

Hit the structure of target plasmid pREPO15

Making up pREPO15 at first is by pcr amplification CMV promotor and hGH exons 1 to be merged.Amplification is from the 1.6 kb fragments of hGH expression construct pXGH308, the CMV promoter region that it has starts from the Nucleotide 546 of gene pool (Genbank) sequence HS5MIEP, finally Nucleotide 2105, utilize oligonucleotide 20 and 35 with said gene pool sequence HS5MIEP and Nucleotide 5225 that starts from gene pool sequence HUGHCSA and the hGH sequence fusion of Nucleotide 7322 finally.Oligonucleotide 20 (35 bp, SEQ ID NO:18) is hybridized in-614 places and CMV promotor with respect to cap site (among the gene pool sequence HEHCMVP1), and comprises a SalI site at its 5 ' end.Oligonucleotide 35 (42 bp, SEQID NO:19) is annealed with the CMV promotor at+966 places with in abutting connection with the hGH exons 1, and comprises preceding 10 base pairs (containing splicing-donor site part) and a HindIII site of hEPO introne 1 at its 5 ' end.With the PCR fragment of HindIII and SalI digestion gained, and use gel-purified.With XhoI and HindIII digested plasmid pT163 (embodiment 2), contain about 1.1 kb fragments of neo ceneme with gel-purified.1.6 kb CMV promotors/hGH exons 1/splicing-donor site fragment and 1.2 kb neo fragments are coupled together, and be inserted into pBSIISK+ (Str-agene, HindIII site Inc).The middle interstitial granules of gained (called after pBNCHS) contain one on transcriptional orientation with CMV promotor/relative neo ceneme of hGH exons 1/segmental direction of splicing-donor site.Make up second middle interstitial granules pREPO5. Δ HindIII, at first digest pREPO5 with HindIII.This process discharges 1.9 kb and two fragments of 8.7kb, and contains the 8.7kb fragment that EPO hits target sequence with gel-purified, connects and cyclisation by self.The plasmid pREPO5 Δ HindIII of gained only contains the non-encoding gene group dna sequence dna that is present in the hEPO upstream region of gene usually.Its included sequence is from respect to-5785～-1 of EPO exons 1.From pBNCHS, excise the 2.8 kb fragments that contain neo, CMV promotor, hGH exons 1 and splicing-donor site with HindIII, and use gel-purified.This fragment through the Klenow of dna polymerase i fragment (New ε ngland Biolabs Inc.) handles and becomes tack, and be connected through BglII digestion and by the pREPO5 Δ HindIII of tack.Cut-1779 bp positions of hEPO exons 1 upstream in pREPO5 Δ HindIII with BglII.The construct pREPO15 (Fig. 8) of gained contains with respect to the sequence of EPO upstream sequence, neo ceneme, CMV promotor, hGH exons 1, splicing-donor site and the hEPO upstream of coding region of hEPO coding region from-5786～-1779 from-1778～-1, each element with listed order according to 5 ' → 3 ' direction assembling with respect to hEPO upstream nucleotide sequence.For transfection people cell, hit the target fragment to discharge 8.6 kb with NotI and PvuI digestion pREPO15.This hits the target fragment and contains that first and second hits target sequence with 4.0 kb respectively of hEPO upstream region of gene dna homology and 1.8 kb.

Cultivation and transfectional cell also identify that the quilt of expressing EPO hits the target clone

All cells maintain 37 ℃, 5%CO ₂, 98% humidity and containing under the condition of DMEM (DMEM/10, HyClone Laboratories) of 10% bovine serum.With 100 μ g DNA electroporation under the condition of 250V and 960 μ F in PBS (GIBCO) 12 * 10 ⁶Cell and finish transfection to secondary human foreskin fibroblast.With the cell handled with every 150mm plate 1 * 10 ⁶The density inoculation of cell second day, is replaced by substratum the DMEM/10 that contains 0.8mg/mlG418 (GIBCO).Chosen process was carried out 14 days, and this moment, resampling was analyzed the EPO output in the substratum.With determining to manifest significant hEPO content (＞5mU/ml) all colonies, and transferring in each hole of 96 well culture plates through EPO ELISA (Genzyme Inc.) on aseptic glass clone post (Bellco) the separation and Culture plate.Cultivate after 1～2 day the sampling output of elisa assay hEPO in these holes.Production hEPO cell strain through cultivating the breeding gained is in order to frozen, separate nucleic acid and the quantitative usefulness of EPO output.

The transfection of HT 1080 cells (ATCC CCL 121) is by handling 12 * 10 among the PBS (GIBCO) with 45 μ g DNA under the condition of 450V and 250 μ F ⁶Cell carries out.Clone's growth is handled secondary human foreskin fibroblast as above-mentioned with identifying.Separate the cloned cell line that produces hEPO with limiting dilution assay.At first the colony that will collect from the initial option flat board is laid on each hole of 24 orifice plates with the density in 10～15 colonies/hole.That will produce hEPO then compiles thing to produce cell density bed board in 96 orifice plates in＜1 colony/hole.Breed each clone to carry out the above-mentioned further analysis the same to human foreskin fibroblast.

Express EPO clone's feature

PRE015 does not contain any hEPO encoding sequence.At hEPO exons 1 upstream neo/CMV promotor/segmental target that hits of hGH exons 1/splicing-donor, express by begin to produce hEPO from the CMV promoter transcription, produce 1.8 kb that comprise CMV sequence, hGH exons 1 and splicing-donor site, hEPO sequence upstream and normal hEPO exon, intron and 3 ' non-primary transcript of translating sequence.Splice above-mentioned transcript to next ortho position in the downstream of hEPO exon 2 splicing-acceptor site from the splicing-donor site of adjacency hGH exons 1.This will produce a new intron of being made up of hEPO upstream region of gene genome sequence, normal hEPO promotor, hEPO exons 1 and hEPO introne 1 effectively.In sophisticated transcript, the hGH exons 1 will substitute the hEPO exons 1.Only encode preceding 4 1/3 amino acid of 26 amino acid signal peptides of hEPO exons 1, it before being gone out by emiocytosis in the past on the body protein cracking fall.Preceding 3 1/3 amino acid of hGH exons 1 coding hGH signal peptide, it went out before being gone out by emiocytosis falls cracking in the past body protein.Replace in the information translation process of hEPO exons 1 at the hGH exons 1, will therefore produce the chimeric protein that a kind of signal peptide wherein is hGH and hEPO sequence.Remove signal peptide by normal post-translation cleavage process and will produce a sophisticated hEPO molecule, its elementary sequence is difficult to separate with normal product.

PRPO015 is transfected into the human fibroblasts causes these cell expressings EPO.Table 5 has been listed with pREPO15 and hit the target result of experiment in human fibroblasts and HT 1080 cell.The hit target frequency of discovery in the normal people inoblast is 1/264 G418 ^rColony, and be 1/450 G418 with the target frequency of hitting of HT 1080 cells ^rColony.Quantitative analysis derives from the hEPO output of each above-mentioned cell strain.The secretory volume that produces gained in the strain at a hEPO who is obtained by transfection people fibrocyte is 7,679mU/10 ⁶Cell/sky (table 5).Output from the activation hEPO clone of HT 1080 cells is 12,582mU/10 ⁶Cell/sky (table 5).These results show, are effectively to the activation of hEPO locus, and cause hEPO constantly to produce with high relatively level.Confirm to hit the target process with Restriction Enzyme and Southern hybridization analysis method and betided the EPO locus.

Human fibroblasts and HT 1080 clones that hit target with pREPO15 are carried out the Southern engram analysis.Fig. 9 A represents parent and is hit the restricted figure of the hEPO locus of target, and Fig. 9 B represents the human fibroblasts clone who is hit target is carried out the result of Restriction Enzyme and Southern hybridization analysis.As the result who hits target at the hEPO locus, the digest of BglII/EcoRI and BamHI discharges the fragment (T1 swimming lane) of 5.8kb and 6.6kb respectively.These two fragments derive from the insertion of the 2.7 kb DNA that contain neo gene and CMV promoter sequence.Because have only a quilt to hit target in two hEPO allelotrope, so in these bacterial strains and mother body D NA, observe the 4.3 kb fragments (BglII/EcoRI) or the 10.6 kb fragments (BamHI) (swimming lane HF) of the unaltered hEPO locus of reflection.These result's proofs cause instructing the new transcription unit that produces human erythropoietin to produce in the homologous recombination that the hEPO locus takes place.Oligonucleotide sequence 20 5 ' TTTTCTCGAG TCGACGACAT TGATTATTGA CTAGT

(SEQ?ID?NO：18) 35 5′TTTTAAGCTT?GAGTACTCAC CTGTAGCCAT GGTGGATCCC?GT

(SEQ?ID?NO：19)

Transfected processed of the activation that the transfection of table 5 pREPO15 in people's cell and hEPO express ^aG418 ^{r b}The cell colony that contains EPO table cell type reaches the dull and stereotyped human fibroblasts 3.3 * 10 of son ⁷264 1HT, 1080 cells 3.1 * 10 ⁷2700 6hEPO express subnumber hEPO and express subnumber ^cThe expression of hEPO/processed cell/processed cell (mU/10 ⁶Cell/24 hour) 1/,264 1/3.3 * 10 ⁷7,679 1/,450 1/5.2 * 10 ⁶12,582

A is by calculating 2 colony number, average its results and be extrapolated to total number of plates and estimate on the flat board.

B is from containing G418 ^rThe EPO elisa assay is carried out in sampling in the substratum of colony flat board, and those demonstrate quilt that the hEPO level is higher than 5mU/ml and can be regarded as EPO and activate phenomenon.

The c quantitative assay is from the hEPO output of human fibroblasts's strain, HF 342-15 or HT 1080 clone HTPREPO15-1-6-6.

Embodiment 8 is by the upstream 1.8kb CMV promotor of the inserting genome hEPO coding region hEPO fusion gene that produces and increase

Structure hits target plasmid pREPO18:

By inserting the dhfr ceneme and make up pREPO18 (Figure 10) being positioned at the neo gene 5 of pREPO15 ' end ClaI site.In order to obtain the dhfr ceneme, with EcoRI and SalI digested plasmid construct pF8CIS9080[Eaton et al., Biochemistry25:8343-8347 (1986)].Purifying contains the 2kb fragment of dhfr ceneme from this digest, and makes it tackization by handling with the Klenow fragment of dna polymerase i.(New England Biolabs Inc.) is connected on the dhfr fragment of tackization with the ClaI joint then.Digest the product of above-mentioned connection again with ClaI, and be connected with the pREPO15 of ClaI digestion.The five equilibrium of this connector is transformed into E.coli, and selects bed board on the flat board at penbritin.Analyze the dhfr segmental direction of bacterium colony by the digestion of Restriction Enzyme to determine to insert.There is its transcriptional orientation plasmid relative of dhfr to be named as pREPO18 (-) with neo gene transcription direction.The second kind of plasmid that has dhfr and neo gene to have identical transcriptional orientation is named as pREPO18 (+).

Cell cultures, transfection also identifies that the quilt of expressing EPO hits the target clone

All cells are maintained at 37 ℃, 5%CO ₂, 98% humidity and containing under the condition of DMEM (DMEM/10, HyClone Laboratories) of 10% bovine serum.Handling in PBS (GIBCO) processing 12 * 10 through electroporation with 45 μ g DNA under 450V and the 250 μ F conditions ⁶Cell and carry out transfection to HT 1080 cells (ATCC CCL 121).With 1 * 10 ⁶The density inoculation of cell/150mm flat board is through the cell of above-mentioned processing.Second day, substratum is replaced by the DMEM/10 that contains 0.8mg/ml G418 (GIBCO).Chosen process was carried out 14 days, the output of hEPO in this sampling analysis substratum in period.Analyze through hEPOELISA (Genzyme Inc.) with trypsin treatment and to be defined as having obvious hEPO output (＞5mU/ml) flat board, and cell carried out again bed board with separating clone.With limiting dilution assay separation of produced hEPO cloned cell line, at first be that the density with 10～15 colonies/hole is laid on the clone in the hole of 24 orifice plates, then with the cell density in＜1 colony/hole in the future the self-produced hEPO cell that compiles thing be plated in 96 orifice plates.Cultivate the usefulness of each clone of breeding in order to frozen, separate nucleic acid and the quantitative analysis of hEPO output.

Select to separate the cell that contains amplification dhfr sequence by methotrexate

The quilt that will produce hEPO after with the pREPO18 transfection hits target G418 ^rClone is with various cell density bed boards, to select in methotrexate (MTX).When after selecting, new clone occurring, then it is measured hEPO output with certain MTX concentration, and in higher MTX concentration (being generally the twice of original content) with various cell densities bed board again.Repeat said process till reaching required hEPO output.In the MTX in each step resistance was selected, DNA isolation and RNA carried out Southern and Northern engram analysis respectively.

Express the clone's of EPO feature:

With pREPO18 transfection HT 1080 cells with two kinds of different dhfr directions.Before the transfection, digest pREPO18 (+) and pREPO18 (-) with XbaI, discharge one 7.9 kb and hit the target fragment, it contains the 2.1 kb districts (with respect to hEPO ATG initiator codon from-3891～-1779) of hEPO exons 1 upstream gene group DNA successively, the 2 kb districts of containing the dhfr gene, the 1.1 kb districts that contain the neo gene, the 1.5kb district of containing the CMV promotor that merges with the hGH exons 1, the 1.1 kb districts of the hEPO introne 1 (containing splicing-donor site) of 10 bp and hEPO exons 1 upstream gene group DNA (with respect to EPO ATG initiator codon from-1778～-678).Derive from two in the experiment transfection and hit the target frequency and be shown in Table 6.By being separated to 5 elementary G418 in these experiments ^rThe clone.They are bred to be used for the quantitative analysis (table 7) that hEPO expresses through substratum.Because pREPO18 contains the dhfr gene, so might be as select to contain the cell of the amplification copy that hits the target construct as described in the embodiment 6 with MTX.Be driven into the G418 of target with what Restriction Enzyme and Southern hybridization analysis confirmed at the hEPO locus ^rThe clone selects by the described substep that carries out in MTX.

Table 6 pREPO18 hits the cell G418 that target construct dna digestion is subject to processing in HT 1080 cells ^rColony pREPO18 XbaI 3.6 * 10 ⁶16,980 (-) pREPO18 XbaI 3.6 * 10 ⁶19,290 (+) have hEPO to express the flat board/G418 of the analyzed son of hEPO expressor ^rColony is time cloning 39 1/,435 1 41 1/,470 4 just

The hEPO that table 7 hits with pREPO18 in HT 1080 clones of target produces

Clone construct hEPO mU/10 ⁶Cell/sky

18B3-147 pREPO18 (+) 24759

18B3-181 pREPO18 (+) 20831

18B3-145 pREPO18 (+) 17586

18B3-168 pREPO18 (+) 5293

18B3-119 pREPO18 (-) 2881

Embodiment 9 in permanence people cell, activate and increase endogenous alpha-interferon, GM-CSF, G-CSF and FSH β gene

Utilize method of the present invention and DNA construct, the endogenous cytogene of many kinds can activate and increase.The general strategy of activation and amplification human's (LeIF), GM-CSF (granulocyte/macrophage colony stimulating factor), G-CSF (granulocyte colony-stimulating factor) and FSH β (follicle-stimulating hormone β subunit) gene is described below.

Alpha-interferon

Human's gene (gene pool sequence HUMIFNAA) coding contains 188 amino acid precursor albumen of 23 amino acid signal peptides.This gene does not contain intron.Figure 11 illustrates a kind of strategy that activates alpha-interferon genes.The designed target construct that hits comprises hitting target sequence, the marker gene that can increase, selectable marker gene, regulatory region, CAP site, splicing-donor site, intron, a splicing-acceptor site with this gene upstream sequence homologous first and being equivalent to first and hits second of target sequence downstream sequence and hit target sequence.For fear of unwanted ATG initiator codon, second hits target sequence will should not extend to than with respect to-107 of normal initiator codon upstreams more.

In described strategy, first and second hit target sequence is hitting in the target gene next-door neighbour mutually normally, but this is not to be essential (vide infra).But this paper has described the marker gene that can increase and has been applicable to the selected marker gene of selection.The marker gene that can increase can be identical gene with selectable marker gene, and its position can be put upside down, and one of them or both can place the intron that hits the target construct.Selectable marker gene is optional, and that the marker gene that can increase only is only when needs carry out amplified reaction is essential.Mixing also of specific C AP site chosen wantonly.Randomly, in hitting the target construct, can comprise that from the exon sequence of another gene 3 ' end to splicing-acceptor site and 5 ' end hits target sequence to second.Regulatory region, CAP site, splicing-donor site, intron and acceptor splicing site site can be used as complete unit and extend the factor-1 α (EF-1 α from the people; Gene pool sequence HUMEF1A) gene or cytomegalovirus (CMV; Gene pool sequence HEHCMVP1) nestle up early stage differentiation from coming out, perhaps mentioned component is by assembling from the isolating suitable component of different genes.

The genomic dna that utilizes recombinant DNA method well known by persons skilled in the art will be equivalent to the alpha-interferon genes upstream can be used as and hits target sequence and hit the assembling of target construct and carry out.As described herein, many selectable and marker gene that can increase can be used for hitting the target construct, and activation and amplified reaction can carry out in many cell types effectively.Utilize embodiment 4 described methods, utilize the ELISA method to measure human's method (BiosourceInternational, Camarillo, CA) can finish to the transfection of elementary, secondary or permanence people cell and to expressing the somatic separation of homologous recombination of alpha-interferon, in addition, can identify the homologous recombination somatocyte by embodiment 1g and the described PCR sieve method of 1j.The cell that contains the amplification copy of the alpha-interferon genes position seat that can increase marker gene and be activated as separation as described in the embodiment 6.

Produced the mRNA precursor in the homologous recombination somatocyte, it comprises that external source exon, splicing-donor site, intron, splicing-acceptor site, second hit target sequence and human coding region and 3 ' non-sequence (Figure 11) of translating.Above-mentioned information is spliced, can be translated to produce human's functional mRNA with producing.

The position that the size of intron reaches with respect to the regulatory region of gene coding region is variable, so that regulatory region performance optimal function.In hitting the target construct, can there be a plurality of exons.In addition, second hits target sequence must not be in close proximity to first and hit target sequence or in its vicinity in normal gene, like this, can leave out the normal upstream of portion gene when carrying out homologous recombination.

GM-CSF

Human GM-CSF gene (gene pool sequence HUMGMCSFG) coding contains 144 amino acid precursor protein of 17 amino acid signal peptides.This gene contains 4 exons and 3 introns, and 50 amino acid of the N of precursor end are coded in first exon, and Figure 12 illustrates the strategy that activates the GM-CSF gene.In this strategy, hit the target construct and be designed to comprise that upstream sequence homologous first with the GM-CSF gene hits target sequence, marker gene that can increase, selectable marker gene, regulatory region, CAP site, coding and is equal to or is equivalent to exon, a splicing-donor site of preceding 50 amino acid whose aminoacid sequences of GM-CSF and hit second of target sequence downstream sequence corresponding to first and hit target sequence on function.Utilize above-mentioned strategy, the mRNA precursor that the homologous recombination somatocyte produces is equivalent to external source exon and splicing-donor site, second and hits target sequence, second and hit any sequence between the initiator codon of target sequence and GM-CSF gene and exon, the intron and the 3 ' non-district (Figure 11) that translates of GM-CSF gene.Above-mentioned information is spliced the exon 2 and the external source exon that cause endogenous GM-CSF gene merge, they produce GM-CSF when being translated.

In above-mentioned strategy, first and second hit target sequence normally hit in the target gene tightly adjacent, but this and nonessential (vide infra).But this paper has described the marker gene that can increase and has been applicable to the selected marker gene of selection.The marker gene that can increase can be identical gene with selectable marker gene, and perhaps its position can be put upside down.Selectable marker gene is optional, and that the marker gene that can increase only is only when needs carry out amplified reaction is essential.Selectable marker gene and/or the marker gene that can increase can be positioned to hit that splicing-donor site and second hits between the target sequence in the target construct.Mixing of specific C AP site is optional.Regulatory region, CAP site and splicing-donor site can be used as complete unit and extend the factor 1 α (EF-1 α from the people; Gene pool sequence HUMEF1A) gene or cytomegalovirus (CMV; Gene pool sequence HEHCMVP1) near separating in the early stage district, perhaps these compositions can be by assembling from the isolating suitable component of different genes (as mMT-1 promotor and CAP site and derive from hGH or the exons 1 of hEPO gene and splice donor site).

Other for example can utilize method, and first and second hit target sequence can be equivalent to sequence in GM-CSF gene first intron.In addition, can utilize and be similar to the described target construct that hits of alpha-interferon, wherein, hitting the target construct is designed to comprise hit target sequence, the marker gene that can increase, selectable marker gene, regulatory region, CAP site, splicing-donor site, intron, a splicing-acceptor site with GM-CSF gene upstream sequence homologous first and be equivalent to first and hits second of target sequence downstream sequence and hit target sequence.

Under any circumstance second hit target sequence and all needn't be close to first in the normal gene and hit target sequence or in its vicinity, like this, the normal upstream part of gene will be deleted when carrying out homologous recombination.In addition, in hitting the target construct, can there be a plurality of non-codings or coding exon.Utilize recombinant DNA well known by persons skilled in the art, to the upstream that is equivalent to the human GM-CSF gene or the gene DNA that includes the subarea as hitting target sequence and the target construct is hit in assembling.As described herein, many selectable and marker gene that can increase can be used to hit in the target construct, and activation process can be carried out in many cell types effectively.By embodiment 4 described methods, use ELISA to measure human GM-CSF method (R﹠amp; D Systems.Minneapolis, MN) can implement elementary, secondary or permanence people cell transfecting with separate the homologous recombination somatocyte of expressing GM-CSF.In addition, utilize above-mentioned PCR sieve method can identify the homologous recombination somatocyte.The cell that separates the amplification copy that contains can increase marker gene and activated GM-CSF gene position seat can be undertaken by preceding method.

G-CSF

Human G-CSF gene (gene pool sequence HUMGCSFG) coding contains 204～207 amino acid precursor protein of 30 amino acid signal peptides.This gene contains 5 exons and 4 introns.13 amino acid of first exons coding signal peptide.Figure 13 illustrates the strategy that activates the G-CSF gene.Hitting the target construct is designed to comprise with G-CSF gene upstream sequence homologous first and hits preceding 13 aminoacid sequences exon identical or aminoacid sequence of equal value on function of target sequence, marker gene that can increase, selectable marker gene, regulatory region, CAP site, coding and G-CSF signal peptide, splicing-donor site and hit target sequence with first downstream sequence corresponding second that hits target sequence.By means of above-mentioned strategy, the homologous recombination somatocyte produces a mRNA precursor, it is corresponding to external source exon and splicing-donor site, second hit target sequence, G-CSF gene start codon and second hits any sequence between target sequence, and the exon of G-CSF gene, intron and the 3 ' non-district (Figure 13) that translates.Above-mentioned information is spliced the exon 2 that causes exogenous exon and endogenous G-CSF gene merge, they will produce G-CSF when being translated.Can carry out functional replacement to preceding 13 amino acid of normal G-CSF signal peptide with the sequence that is present in the exogenous exon, this just makes anyone to modify signal peptide, and therefore proteinic secretion characteristic produces.

In above-mentioned strategy, first and second hit target sequence hits in the target gene normally and is close to, but this and inessential.Second hits target sequence needn't be in close proximity to first and hit target sequence or in its vicinity in normal gene, like this, leave out the normal upstream part of gene when carrying out homologous recombination.The marker gene that can increase can be identical gene with selectable marker gene, and perhaps its position can be reversed.Selectable marker gene is optional, and the marker gene that can increase is only just essential when needs carry out amplified reaction.Selectable marker gene and/or the marker gene that can increase can be positioned at the splicing-donor site and second that hits the target construct and hit between the target sequence.Mixing of specific C AP site is can be elective.Regulatory region, CAP site and splicing-donor site can be used as complete unit and extend the factor-1 α (EF-1 α from the people; Gene pool sequence HUMEF1A) gene or cytomegalovirus (CMV; Gene pool sequence HEHCMVP1) near separating in the early stage district, perhaps these compositions can be by assembling from the isolating suitable component of different genes (as mMT-I promotor and CAP site, deriving from the exons 1 and the splicing-donor site of hGH or EPO gene).In hitting the target construct, can use a plurality of codings or noncoding external source exon, as long as when splicing, be present in an ATG initiator codon in the sign indicating number that mature protein is arranged and be included in one of them exon and get final product.

Other available method, for example first and second sequences of hitting in first intron that target sequence can be equivalent to the G-CSF gene.In addition, can use be similar to described alpha-interferon hit the target construct, wherein, hitting the target construct is designed to comprise: hit target sequence, one with G-CSF gene upstream sequence homologous first and can expand marker gene, selectable marker gene, regulatory region, CAP site, splicing-donor site, intron, a splicing-acceptor site of grave and hit second of target sequence downstream sequence corresponding to first and hit target sequence.

Use recombinant DNA method well known by persons skilled in the art, can with corresponding to the upstream of human G-CSF gene or the genomic dna that includes the subarea as hitting target sequence and can implementing to the assembling of hitting the target construct.As described herein, in hitting the target construct, can use many marker gene of selecting and can increasing, and activation process goes out and can carry out effectively in many cell types.Utilize embodiment 4 described methods, use ELISA to measure human G-CSF method (R﹠amp; DSystems, Minneapolis MN) can carry out the transfection of elementary, secondary or permanence people cell and somatic separation of homologous recombination of expression G-CSF, in addition, also can identify the homologous recombination somatocyte by means of aforesaid PCR screening.The cell that separates the amplification copy that contains the alpha-interferon genes position seat that can increase marker gene and be activated with preceding method.

FSHβ

People FSH β gene (gene pool sequence HUMFSH1) coding contains 129 amino acid precursor protein of 16 amino acid signal peptides.This gene contains three and each and every one shows son and two introns, and wherein first exon is the exon of non-coding.Can reach activation with multiple strategy to FSH β.Figure 14 illustrates a kind of activation strategy.In this strategy, hit the target construct and be designed to comprise and suitable hit target sequence, the marker gene that can increase, selectable marker gene, regulatory region, CAP site, exon, a splicing-donor site with FSH β gene upstream sequence homologous first and hit second of target sequence downstream sequence corresponding to first and hit target sequence.By means of above-mentioned strategy, the homologous recombination somatocyte produces a mRNA precursor, it is equivalent to external source exon and splicing-donor site, second and hits any sequence between target sequence, second target sequence and the FSH β gene start codon, and the exon of FSH β gene, intron and the 3 ' non-district (Figure 14) that translates.Above-mentioned information is spliced the exon 2 that will cause external source exon and endogenous FSH β gene merge, they can produce FSH β when being translated.In above-mentioned strategy, first and second hit target sequence is close to normally hitting in the target gene, but this and nonessential (vide infra).

Other available method, for example first and second hit target sequence can be corresponding to the sequence in FSH β gene first intron.In addition, can use and be similar to the described target construct that hits of alpha-interferon.In this strategy, hit the target construct and be designed to comprise and hit target sequence, the marker gene that can increase, selectable marker gene, regulatory region, CAP site, splicing-donor site, intron, an acceptor splicing site site with FSH β gene upstream sequence homologous first and hit second of target sequence downstream sequence corresponding to first and hit target sequence.Second hits target sequence should not extend to than with respect to-40 of normal FSH β transcription initiation site upstreams more, to avoid unwanted ATG initiator codon.In the homologous recombination somatocyte, the mRNA precursor that is produced comprises that external source exon, splicing-donor site, intron, splicing-acceptor site, second hit target sequence and people FSH β coding exon, intron and 3 ' do not translate sequence, these information are spliced will produce the functional mRNA that can be translated to production people FSH β.The size of intron and the regulatory region that therefore produced are variable with respect to the position of gene coding region, so that regulatory region performance optimal function.

In any one activation strategy, second hits target sequence needn't be arranged in first of next-door neighbour's normal gene and hit target sequence or in its vicinity, like this, when carrying out homologous recombination, normal upstream part that will the deletion gene.And one is hit the upstream that target sequence can be a gene, and one is hit in the exon or intron that target sequence may reside in FSH β gene.

The marker gene that can increase can be identical gene with selectable marker gene, and its position can be put upside down, and one of them or both can be present in the intron that hits the target construct.This paper has described marker gene that can increase and the selectable marker gene that is suitable for selecting.Selectable marker gene is optional, and that the marker gene that can increase only is only when needs carry out amplified reaction is essential.Mixing of specific C AP site is can be elective.Randomly, can be included in from the exon sequence of another gene and hit target construct 3 ' to splicing-acceptor site and 5 ' hit target sequence to second.Regulatory region, CAP site, exon, splicing-donor site, intron and splicing-acceptor site can be used as complete unit and extend the factor-1 α (EF-1 α from the people; Gene pool sequence HUMEF1A) gene or cytomegalovirus (CMV; Gene pool sequence HEHCMVP1) near separating in the early stage district, perhaps these compositions are assembled by isolating suitable component from different genes.Under any circumstance, the external source exon can be identical or different with first exon of normal FSH β gene, and can have a plurality of exons in hitting the target construct.

Utilize recombinant technology well known by persons skilled in the art, the genomic dna corresponding to FSH β upstream region of gene district can be able to be assembled as hitting target sequence and hitting the target construct.As described herein, many selectable and marker gene that can increase can be used to hit in the target construct, and activation process can be carried out in many cell types effectively.If necessary, can produce the FSH β gene product that is activated in the cell type of expressing human glycoprotein alpha subunit, the product of glycoprotein alpha subunit and FSH β gene product form heterodimer.This can be a naturally occurring cell strain or clone.In addition, human glucoprotein alpha subunit gene (gene pool sequence HUMGLYCA1) can with FSH β gene product co expression.And above-mentioned co expression is finished by human glucoprotein alpha subunit gene or cDNA under suitable promoter regulation, or is finished by the activation of human glucoprotein alpha subunit by methods described herein.Utilize preceding method use ELISA measure people FSH β (Accurate Chemicaland Scientific, Westbury, NY) can carry out to the transfection of elementary, secondary or permanence people cell with to expressing somatic separation of homologous recombination of FSH β.In addition, utilize aforementioned PCR sieve method also can identify the homologous recombination somatocyte.The cell that separates the amplification copy that contains marker gene that can increase and the alpha-interferon genes position seat that is activated as previously mentioned.

Person of skill in the art will appreciate that, maybe can utilize not transnormal experiment and can determine many Equivalents of the specific embodiments of invention described herein.This class Equivalent will be included by claims.

Claims (132)

1. DNA construct changes intracellular target gene expression during its target site homologous recombination in DNA construct and cell chromosome DNA, and DNA construct comprises:

(a) with target site homologous targeting sequencing;

(b) external source is regulated sequence;

(c) exon;

(d) the unpaired donor splicing site of the unpaired 3 ' end of exon;

Wherein, after targeting sequencing and the target site homologous recombination, except giving birth in all of target gene the exon, the chromosomal DNA of cell also comprises construct-deutero-exon, and wherein the protein chosen from following group of target gene coding comprises gamma-interferon, interleukin 1, interleukin II, interleukin, interleukin 6, interleukin-11, interleukin 12, GSF-granulocyte (G-CSF), CSF-scavenger cell (M-CSF), CSF-granulocyte/scavenger cell (GM-CSF), FSH β, TGF-β, tumour necrosis factor, hyperglycemic-glycogenolytic factor, bone growth factor-2, bone growth factor-7, TSH-β, protein kinase C, urokinase, Antithrombin III, DNA-se, alpha-galactosidase, tyrosine hydroxylase, blood coagulation factor V, blood coagulation factor VII, blood coagulation factor X, blood coagulation factor XI, plasma thromboplastin antecedent II, IL-2 antagonist and solvable CD4.

2. according to the DNA construct of claim 1, wherein construct derive exon comprise with target gene first in give birth to the different dna sequence dna of dna sequence dna of exon.

3. DNA construct during its target site homologous recombination in DNA construct and cell chromosome DNA, changes intracellular target gene expression, and DNA construct comprises:

(a) with target site homologous targeting sequencing;

(b) external source is regulated sequence;

(c) exon;

(d) at exon 3 ' terminal unpaired donor splicing site;

Wherein, after targeting sequencing and target site homologous recombination, (b)-(d) be positioned at the upstream of the transcription initiation site of target gene, wherein the protein chosen from following group of target gene coding comprises gamma-interferon, interleukin 1, interleukin II, interleukin, interleukin 6, interleukin-11, interleukin 12, GSF-granulocyte (G-CSF), CSF-scavenger cell (M-CSF), CSF-granulocyte/scavenger cell (GM-CSF), FSH β, TGF-β, tumour necrosis factor, hyperglycemic-glycogenolytic factor, bone growth factor-2, bone growth factor-7, TSH-β, protein kinase C, urokinase, Antithrombin III, DNA-se, alpha-galactosidase, tyrosine hydroxylase, blood coagulation factor V, blood coagulation factor VII, blood coagulation factor X, blood coagulation factor XI, plasma thromboplastin antecedent II, IL-2 antagonist and solvable CD4.

4. according to the DNA construct of claim 3, wherein exon comprise with target gene first in give birth to the different dna sequence dna of dna sequence dna of exon.

5. according to the DNA construct of claim 3, target gene coding alpha-galactosidase wherein.

6. according to the DNA construct of claim 3, wherein construct deutero-exon is identical with first exon of FSH β gene.

7. according to the DNA construct of claim 6, the cell expressing human glycoprotein α-subunit that is wherein obtained.

8. according to the DNA construct of claim 6, cell expressing human class glycoprotein α-subunit under the control of exogenous promoter wherein.

9. according to the DNA construct of claim 3, expression of target gene GM-CSF wherein.

10. according to the DNA construct of claim 9,50 amino acid at first of construct deutero-exons coding GM-CSF precursor protein wherein.

11. according to the DNA construct of claim 9, wherein construct deutero-exons coding comprises the sequence of sense signal peptide.

12. according to the DNA construct of claim 3, target gene coding G-CSF wherein.

13. according to the DNA construct of claim 12,13 amino acid at first of construct deutero-exons coding G-CSF signal peptide wherein.

14. a DNA construct during its target site homologous recombination in DNA construct and cell chromosome DNA, changes intracellular target gene expression, DNA construct comprises:

(a) with target site homologous targeting sequencing;

(b) external source is regulated sequence;

(c) exon;

(d) exon 3 ' terminal not pairing montage-donor site;

Wherein, after targeting sequencing and target site homologous recombination, (b)-(d) be positioned at the upstream of the transcription initiation site of target gene, wherein external source adjusting sequence is the adjusting sequence of adenoviral gene, the adjusting sequence of collagen gene, the adjusting sequence of actin gene, the adjusting sequence of HMG-CoA reductase gene, or the adjusting sequence of EF-1 α gene.

15. a method that changes expression of target gene in the cell, method comprises step:

(a) provide cell, its genome comprises:

(i) contain interior target gene of giving birth to regulation domain;

(ii) target site;

(b) provide DNA construct to comprise:

(i) with target site homologous targeting sequencing;

(ii) external source is regulated sequence;

(iii) exon;

(iv) exon 3 ' terminal unpaired donor splicing site;

(c) use the DNA construct transfectional cell, thereby produce transfectional cell;

(d) transfectional cell is remained under the condition that is suitable for homologous recombination, thereby produce the homologous recombination cell, give birth to the exon except in all of target gene, its genome comprises that external source regulates sequence, construct exon and the construct donor splicing site of deriving of deriving;

(e) the homologous recombination cell is remained under the condition that is suitable for transcribing, regulate in external source under the control of sequence, produce the construct exon of deriving, target gene and at the derive transcription of any sequence between exon and the target gene of construct, wherein corresponding to construct derive the transcription RNA of donor splicing site with in target gene corresponding to the directed montage of the acceptor splicing site of the transcription in a site, the protein from following group, chosen of target gene coding wherein, comprise gamma-interferon, nerve growth factor, interleukin 1, interleukin II, interleukin, FSH β, TGF-β, tumour necrosis factor, hyperglycemic-glycogenolytic factor, bone growth factor-2, bone growth factor-7, TSH-β, interleukin 6, interleukin-11, interleukin 12, GSF-granulocyte (G-CSF), CSF-scavenger cell (M-CSF), CSF-granulocyte/scavenger cell (GM-CSF), protein kinase C, urokinase, Antithrombin III, DNA-se, alpha-galactosidase, tyrosine hydroxylase, blood coagulation factor V, blood coagulation factor VII, blood coagulation factor X, blood coagulation factor XI, plasma thromboplastin antecedent II, IL-2 antagonist and solvable CD4.

16., further comprise step according to the method for claim 15:

(f) the homologous recombination cell is remained under the condition that is suitable for transcription montage and translation;

(g) confirmation has produced the translated product of transcription.

17. a method that changes expression of target gene in the cell, method comprises step:

(a) provide cell, its genome comprises:

(i) contain interior target gene of giving birth to regulation domain;

(ii) target site;

(b) provide DNA construct to comprise:

(i) with target site homologous targeting sequencing;

(ii) external source is regulated sequence;

(iii) exon;

(iv) exon 3 ' terminal unpaired donor splicing site;

(c) use the DNA construct transfectional cell, thereby produce transfectional cell;

(d) transfectional cell is remained under the condition that is suitable for homologous recombination, thereby produce the homologous recombination cell, its genome comprises that external source regulates sequence, construct exon and the construct donor splicing site of deriving of deriving, and all are positioned at the upstream of the living transcription initiation site of target gene;

(e) the homologous recombination cell is remained under the condition that is suitable for transcribing, regulate in external source under the control of sequence, produce the construct exon of deriving, target gene and at the derive transcription of any sequence between exon and the target gene of construct, wherein corresponding to construct derive the transcription RNA of donor splicing site with in target gene corresponding to the directed montage of the acceptor splicing site of the transcription in a site, the protein from following group, chosen of target gene coding wherein, comprise gamma-interferon, nerve growth factor, interleukin 1, interleukin II, interleukin, FSH β, TGF-β, tumour necrosis factor, hyperglycemic-glycogenolytic factor, bone growth factor-2, bone growth factor-7, TSH-β, interleukin 6, interleukin-11, interleukin 12, GSF-granulocyte (G-CSF), CSF-scavenger cell (M-CSF), CSF-granulocyte/scavenger cell (GM-CSF), protein kinase C, urokinase, Antithrombin III, DNA-se, alpha-galactosidase, tyrosine hydroxylase, blood coagulation factor V, blood coagulation factor VII, blood coagulation factor X, blood coagulation factor XI, plasma thromboplastin antecedent II, IL-2 antagonist and solvable CD4.

18., further comprise step according to the method for claim 17:

(f) the homologous recombination cell is remained under the condition that is suitable for transcription montage and translation;

(g) confirmation has produced the translated product of transcription.

19. a method that changes expression of target gene in the cell, method comprises step:

(a) provide cell, its genome comprises:

(i) contain interior target gene of giving birth to regulation domain;

(ii) target site;

(b) provide DNA construct to comprise:

(i) with target site homologous targeting sequencing;

(ii) external source is regulated sequence;

(iii) exon;

(iv) exon 3 ' terminal unpaired donor splicing site;

(c) use the DNA construct transfectional cell, thereby produce transfectional cell;

(d) transfectional cell is remained under the condition that is suitable for homologous recombination, thereby produce the homologous recombination cell, its genome comprises that external source regulates sequence, construct exon and the construct donor splicing site of deriving of deriving, and all are positioned at the upstream of the living transcription initiation site of target gene;

(e) the homologous recombination cell is remained under the condition that is suitable for transcribing, regulate in external source under the control of sequence, produce the construct exon of deriving, target gene and at the derive transcription of any sequence between exon and the target gene of construct, wherein corresponding to construct derive the transcription RNA of donor splicing site with in target gene corresponding to the directed montage of the acceptor splicing site of the transcription in a site, wherein external source adjusting sequence is the adjusting sequence of adenoviral gene, the adjusting sequence of collagen gene, the adjusting sequence of actin gene, the adjusting sequence of immunoglobulin gene, the adjusting sequence of HMG-CoA reductase gene, or the adjusting sequence of EF-1 α gene.

20., further comprise step according to the method for claim 19:

(f) the homologous recombination cell is remained under the condition that is suitable for transcription montage and translation;

(g) confirmation has produced the translated product of transcription.

21. the vertebrate cells of a cultivation, in its genome, mix transcription unit, wherein transcription unit comprises external source adjusting sequence, the external source exon, with at the external source exon 3 ' terminal donor splicing site, all are positioned at the upstream of the living transcription initiation site of giving birth to gene, donor splicing site operationally combines with the interior living acceptor splicing site of the second interior life exon of interior living gene, give birth to the protein that genes encoding is chosen wherein from following group, comprise gamma-interferon, nerve growth factor, FSH β, TGF-β, tumour necrosis factor, hyperglycemic-glycogenolytic factor, bone growth factor-2, bone growth factor-7, TSH-β, interleukin 1, interleukin II, interleukin, interleukin 6, interleukin-11, interleukin 12, GSF-granulocyte (G-CSF), CSF-scavenger cell (M-CSF), CSF-granulocyte/scavenger cell (GM-CSF), protein kinase C, urokinase, Antithrombin III, DNA-se, alpha-galactosidase, tyrosine hydroxylase, blood coagulation factor V, blood coagulation factor VII, blood coagulation factor X, blood coagulation factor XI, plasma thromboplastin antecedent II, IL-2 antagonist and solvable CD4.

22. the vertebrate cells of a cultivation, in its genome, mix transcription unit, transcription unit comprises external source adjusting sequence, the external source exon and at the external source exon 3 ' terminal donor splicing site, donor splicing site and interior living gene second in the interior living acceptor splicing site of life exon operationally combine, wherein in giving birth to all in the gene in being present in, give birth to the exon, cell also comprises the external source exon, and the protein that wherein interior living genes encoding is chosen from following group comprises gamma-interferon, nerve growth factor, FSH β, TGF-β, tumour necrosis factor, hyperglycemic-glycogenolytic factor, bone growth factor-2, bone growth factor-7, TSH-β, interleukin 1, interleukin II, interleukin, interleukin 6, interleukin-11, interleukin 12, GSF-granulocyte (G-CSF), CSF-scavenger cell (M-CSF), CSF-granulocyte/scavenger cell (GM-CSF), protein kinase C, urokinase, Antithrombin III, DNA-se, alpha-galactosidase, tyrosine hydroxylase, blood coagulation factor V, blood coagulation factor VII, blood coagulation factor X, blood coagulation factor XI, plasma thromboplastin antecedent II, IL-2 antagonist and solvable CD4.

23. according to the vertebrate cells of the cultivation of claim 22, wherein cell is chosen from following group, comprises the HT1080 cell, the HeLa cell, HeLa cell derivative, MCF-7 breast cancer cell, K-562 leukemia cell, KB cancer cells, 2780AD ovarian cancer cell, the Raji cell, Jurkat cell, Namalwa cell, the HL-60 cell, Daudi cell, RPMI 8226 cells, the U-937 cell, the Bows melanoma cells, the WI-38VA13 subspecies are cell and MOLT-4 cell.

24. vertebrate cells according to the cultivation of claim 22, wherein external source adjusting sequence is the adjusting sequence of adenoviral gene, the adjusting sequence of collagen gene, the adjusting sequence of actin gene, the adjusting sequence of immunoglobulin gene, the adjusting sequence of HMG-CoA reductase gene, or the adjusting sequence of EF-1 α gene.

25. a DNA construct during its target site homologous recombination in DNA construct and cell chromosome DNA, changes intracellular target gene expression, target site is positioned at the upstream of the living transcription initiation site of target gene, and DNA construct comprises:

(a) with target site homologous targeting sequencing;

(b) external source is regulated sequence;

(c) exon;

(d) donor splicing site;

(e) intron;

(f) acceptor splicing site;

Wherein, because homologous recombination, external source is regulated the transcribing of whole encoding sequences that the sequence setting control comprises the sequence of (c)-(f) and target gene.

26., wherein after homologous recombination, (b)-(f) be positioned at the upstream of regulating sequence of giving birth to of target gene according to the DNA construct of claim 25.

27. according to the DNA construct of claim 25, wherein construct further comprises interior upstream sequence homologous second targeting sequencing that give birth to regulate sequence with target gene.

28. according to the DNA construct of claim 25, target gene coding therapeutic protein wherein.

29. according to the DNA construct of claim 25, target gene coding hormone wherein, cytokine, antigen, antibody, enzyme, thrombin, transport protein matter, acceptor is regulated protein, structural protein, or transcription factor.

30. according to the DNA construct of claim 25, wherein the protein chosen from following group of target gene coding comprises regucalin, tethelin, Regular Insulin, pancreotropic hormone, insulin-like growth factor, Rat parathyroid hormone 1-34, alpha-interferon, beta-interferon, gamma-interferon, nerve growth factor, FSH β, TGF-β, tumour necrosis factor, hyperglycemic-glycogenolytic factor, bone growth factor-2, bone growth factor-7, TSH-β, interleukin 1, interleukin II, interleukin, interleukin 6, interleukin-11, interleukin 12, GSF-granulocyte (G-CSF), CSF-scavenger cell (M-CSF), CSF-granulocyte/scavenger cell (GM-CSF), immunoglobulin (Ig), catalytic antibody, protein kinase C, glucocerebrosidase, superoxide dismutase, organize the plasminogen activator, urokinase, Antithrombin III, DNA-se, tyrosine hydroxylase, blood coagulation factor V, blood coagulation factor VII, the blood coagulation Factor IX, the blood coagulation factors IX, blood coagulation factor X, blood coagulation factor XI, plasma thromboplastin antecedent II, apolipoprotein E, apolipoprotein A-I, globin, low density lipoprotein receptor, the IL-2 acceptor, the IL-2 antagonist, α-1 antitrypsin, immune response modifier and solvable CD4.

31. according to the DNA construct of claim 25, target gene coding alpha-galactosidase wherein.

32. according to the DNA construct of claim 25, target gene coding alpha-interferon wherein.

33. according to the DNA construct of claim 25, target gene coding glucocerebrosidase wherein.

34. according to the DNA construct of claim 25, wherein external source adjusting sequence is a promotor, enhanser, skeleton-connecting zone, or transcription factor binding site point.

35., comprise that further second regulates sequence according to the DNA construct of claim 34.

36. according to the DNA construct of claim 34, wherein external source adjusting sequence is the adjusting sequence of adenoviral gene, the adjusting sequence of SV-40 gene, or the adjusting sequence of cytomegalovirus gene.

37. DNA construct according to claim 34, wherein regulating sequence is the adjusting sequence of mouse metallothioneins-I gene, the adjusting sequence of collagen gene, the adjusting sequence of actin gene, the adjusting sequence of immunoglobulin gene, the adjusting sequence of HMG-CoA reductase gene, or the adjusting sequence of EF-1 α gene.

38., further comprise a gene according to the DNA construct of claim 25.

39., further comprise one or more selectable marker gene according to the DNA construct of claim 25.

40., further comprise a marker gene that can increase according to the DNA construct of claim 39.

41. a method that changes expression of target gene in the cellular genome comprises step:

(a) with DNA construct with cell transfecting, DNA construct comprises:

(i) targeting sequencing;

(ii) external source is regulated sequence;

(iii) exon;

(iv) donor splicing site;

(v) intron;

(vi) acceptor splicing site, thus transfectional cell produced;

(b) transfectional cell is remained under the condition that is suitable for homologous recombination, thereby produce

Give birth to the homologous recombination cell, the setting control of external source adjusting sequence comprises (iii)-(transcribing of sequence vi) and target gene encoding sequence in the cell.

(c) the homologous recombination cell is remained under the condition that is suitable for transcribing, regulate in external source under the control of sequence, thereby produce transcription.

42. according to the method for claim 41, wherein exon comprises the CAP site.

43. according to the method for claim 42, wherein exon comprises nucleotide sequence ATG.

44. a fused protein is according to claim 43 preparation and contain by first aminoacid sequence of the exons coding of DNA construct with by second aminoacid sequence of target gene coding.

45. according to the method for claim 41, wherein the upstream sequence homology of sequence is regulated in the interior life of targeting sequencing and target gene.

46. according to the method for claim 41, wherein construct further comprises interior upstream sequence homologous second targeting sequencing that give birth to regulate sequence with target gene.

47. according to the method for claim 41, wherein cell is the human cell.

48. according to the method for claim 41, target gene coding therapeutic protein wherein.

49. according to the method for claim 41, target gene coding hormone wherein, cytokine, antigen, antibody, enzyme, thrombin, transport protein matter, acceptor is regulated protein, structural protein, or transcription factor.

50. according to the method for claim 41, wherein the protein chosen from following group of target gene coding comprises regucalin, tethelin, Regular Insulin, pancreotropic hormone, insulin-like growth factor, Rat parathyroid hormone 1-34, alpha-interferon, beta-interferon, gamma-interferon, nerve growth factor, FSH β, TGF-β, tumour necrosis factor, hyperglycemic-glycogenolytic factor, bone growth factor-2, bone growth factor-7, TSH-β, interleukin 1, interleukin II, interleukin, interleukin 6, interleukin-11, interleukin 12, GSF-granulocyte (G-CSF), CSF-scavenger cell (M-CSF), CSF-granulocyte/scavenger cell (GM-CSF), immunoglobulin (Ig), catalytic antibody, protein kinase C, glucocerebrosidase, superoxide dismutase, organize the plasminogen activator, urokinase, Antithrombin III, DNA-se, tyrosine hydroxylase, blood coagulation factor V, blood coagulation factor VII, the blood coagulation Factor IX, the blood coagulation factors IX, blood coagulation factor X, blood coagulation factor XI, plasma thromboplastin antecedent II, apolipoprotein E, apolipoprotein A-I, globin, low density lipoprotein receptor, the IL-2 acceptor, the IL-2 antagonist, α-1 antitrypsin, immune response modifier and solvable CD4.

51. according to the method for claim 41, target gene coding alpha-galactosidase wherein.

52. according to the method for claim 41, target gene coding alpha-interferon wherein.

53. according to the method for claim 41, target gene coding glucocerebrosidase wherein.

54. according to the method for claim 41, wherein external source adjusting sequence is a promotor, enhanser, skeleton-connecting zone, or transcription factor binding site point.

55., comprise that further second regulates sequence according to the method for claim 54.

56. according to the method for claim 54, wherein external source adjusting sequence is the adjusting sequence of adenoviral gene, the adjusting sequence of SV-40 gene, or the adjusting sequence of cytomegalovirus gene.

57. method according to claim 54, wherein regulating sequence is the adjusting sequence of mouse metallothioneins-I gene, the adjusting sequence of collagen gene, the adjusting sequence of actin gene, the adjusting sequence of immunoglobulin gene, the adjusting sequence of HMG-CoA reductase gene, or the adjusting sequence of EF-1 α gene.

58., further comprise step according to the method for claim 41:

(f) the homologous recombination cell is remained under the condition that is suitable for transcription montage and translation;

(g) confirmation has produced the translated product of transcription.

59. glucocerebrosidase according to claim 58 preparation.

60. the vertebrate cells of a cultivation mixes transcription unit in its genome, wherein transcription unit comprises external source adjusting sequence, the external source exon, donor splicing site, intron, and acceptor splicing site, all all are positioned at the upstream of the living transcription initiation site of giving birth to gene, the setting control of external source adjusting sequence comprises external source exon, donor splicing site, intron like this, transcribing of the sequence of acceptor splicing site and target gene encoding sequence, thus transcription produced.

61. according to the vertebrate cells of the cultivation of claim 60, wherein the external source exon comprises the CAP site.

62. according to the vertebrate cells of the cultivation of claim 60, wherein external source is regulated sequence, external source exon and donor splicing site are in the upstream of the encoding sequence of interior living gene.

63. according to the vertebrate cells of the cultivation of claim 60, interior the giving birth to of giving birth to gene in wherein regulated sequence and lacked.

64. according to the vertebrate cells of the cultivation of claim 60, give birth in wherein gene first in give birth to exon and lack.

65. according to the vertebrate cells of the cultivation of claim 60, give birth to the genes encoding hormone in wherein, cytokine, antigen, antibody, enzyme, thrombin, transport protein matter, acceptor is regulated protein, structural protein, or transcription factor.

66. according to the vertebrate cells of the cultivation of claim 60, give birth to the protein that genes encoding is chosen in wherein from following group, comprise regucalin, tethelin, Regular Insulin, pancreotropic hormone, insulin-like growth factor, Rat parathyroid hormone 1-34, alpha-interferon, beta-interferon, gamma-interferon, nerve growth factor, FSH β, TGF-β, tumour necrosis factor, hyperglycemic-glycogenolytic factor, bone growth factor-2, bone growth factor-7, TSH-β, interleukin 1, interleukin II, interleukin, interleukin 6, interleukin-11, interleukin 12, GSF-granulocyte (G-CSF), CSF-scavenger cell (M-CSF), CSF-granulocyte/scavenger cell (GM-CSF), immunoglobulin (Ig), catalytic antibody, protein kinase C, glucocerebrosidase, superoxide dismutase, organize the plasminogen activator, urokinase, Antithrombin III, DNA-se, tyrosine hydroxylase, blood coagulation factor V, blood coagulation factor VII, the blood coagulation Factor IX, the blood coagulation factors IX, blood coagulation factor X, blood coagulation factor XI, plasma thromboplastin antecedent II, apolipoprotein E, apolipoprotein A-I, globin, low density lipoprotein receptor, the IL-2 acceptor, the IL-2 antagonist, α-1 antitrypsin, immune response modifier and solvable CD4.

67., give birth to the genes encoding alpha-interferon in wherein according to the vertebrate cells of the cultivation of claim 60.

68., give birth to the genes encoding alpha-galactosidase in wherein according to the vertebrate cells of the cultivation of claim 60.

69., give birth to the genes encoding glucocerebrosidase in wherein according to the vertebrate cells of the cultivation of claim 60.

70. a kind of plant or fungic origin culturing cell mix transcription unit in its genome, wherein transcription unit comprises external source adjusting sequence, the external source exon, donor splicing site, intron, and acceptor splicing site, all all are positioned at the upstream of the living transcription initiation site of giving birth to gene, the setting control of external source adjusting sequence comprises external source exon, donor splicing site, intron like this, transcribing of the sequence of acceptor splicing site and target gene encoding sequence, thus transcription produced.

71. according to the vertebrate cells of the cultivation of claim 60, wherein cell is a mammalian cell.

72. according to the vertebrate cells of the cultivation of claim 71, wherein cell is primary or secondary mammalian cell.

73. according to the vertebrate cells of the cultivation of claim 71, wherein cell is primary or secondary human cell.

74. according to the vertebrate cells of the cultivation of claim 60, wherein cell is the immortalization mammalian cell.

75. according to the vertebrate cells of the cultivation of claim 74, wherein cell is the immortalization human cell.

76. according to the vertebrate cells of the cultivation of claim 60, wherein cell is chosen from following group, comprises the HeLa cell, the HeLa cell derivative, MCF-7 breast cancer cell, K-562 leukemia cell, the KB cancer cells, 2780AD ovarian cancer cell, Raji cell, the Jurkat cell, Namalwa cell, HL-60 cell, the Daudi cell, RPMI 8226 cells, U-937 cell, Bows melanoma cells, WI-38VA13 subspecies are cell and MOLT-4 cell.

77. according to the vertebrate cells of the cultivation of claim 60, wherein cell is the HT1080 cell.

78. according to the vertebrate cells of the cultivation of claim 60, cell expressing therapeutic protein wherein, protein is by transcription translation preparation.

79. according to the vertebrate cells of the cultivation of claim 60, wherein to regulate sequence be promotor to external source, enhanser, skeleton-connecting zone, or transcription factor binding site point.

80. according to the vertebrate cells of the cultivation of claim 79, wherein to regulate sequence be the adjusting sequence of adenoviral gene to external source, the adjusting sequence of SV-40 gene, or the adjusting sequence of cytomegalovirus gene.

81. vertebrate cells according to the cultivation of claim 79, wherein external source adjusting sequence is the adjusting sequence of mouse metallothioneins-I gene, the adjusting sequence of collagen gene, the adjusting sequence of actin gene, the adjusting sequence of immunoglobulin gene, the adjusting sequence of HMG-CoA reductase gene, or the adjusting sequence of EF-1 α gene.

82. a method for preparing the homologous recombination cell comprises step:

(a) provide cell, its genome comprises:

(i) contain interior target gene of giving birth to regulation domain;

(ii) in target gene or the target site of upstream;

(b) provide DNA construct, comprising:

(i) with target site homologous targeting sequencing;

(ii) external source is regulated sequence;

(iii) exon;

(iv) donor splicing site;

(v) intron;

(vi) acceptor splicing site,

(c) use the DNA construct transfectional cell, thereby produce transfectional cell;

(d) transfectional cell being remained under the condition that is suitable for homologous recombination, thereby produce the homologous recombination cell, give birth to the exon except in all of target gene, cellular genome comprises (ii)-(vi).

83. homologous recombination cell according to the preparation of the method for claim 82.

84. a method that changes expression of target gene in the cell comprises step:

(a) use the DNA construct transfectional cell, construct comprises:

(i) targeting sequencing;

(ii) regulate sequence;

(iii) exon;

(iv) donor splicing site;

(v) intron;

(vi) acceptor splicing site, thus transfectional cell produced;

(b) transfectional cell is remained under the condition that is suitable for homologous recombination, thereby produce the homologous recombination cell, the setting control of the sequence of external source adjusting therein comprises (iii)-(transcribing of sequence vi) and the whole encoding sequences of target gene;

(c) the homologous recombination cell is remained under the condition that is suitable for the expression of external source adjusting sequence.

85. a method that changes expression of target gene in the cell comprises step:

(a) provide cell, its genome comprises:

(i) target gene;

(ii) in target gene or the target site of upstream;

(b) provide DNA construct, comprising:

(i) with target site homologous targeting sequencing;

The (ii) promotor that obtains from Mammals Actin muscle or collagen gene;

(c) use the DNA construct transfectional cell, thereby produce transfectional cell;

(d) transfectional cell is remained under the condition that is suitable for homologous recombination, thereby produce the homologous recombination cell, the genome of cell contains transcribing of positioning starting control target gene;

(e) the homologous recombination cell is remained under promotor control, be suitable under the condition that target gene transcribes.

86. the vertebrate cells of a cultivation mixes the novel transcription unit of giving birth to genetic transcription in the setting control that contains external source or collagen promotor in its genome, give birth to the gene Actin muscle collagen of also not encoding of neither encoding in wherein.

87. a method that changes expression of target gene in the cell chromosome comprises step:

(a) use the DNA construct transfectional cell, comprising:

(i) with the coding region of target gene in or the genomic dna homologous targeting sequencing of upstream;

(ii) Actin muscle or collagen promotor;

(iii) exon;

(iv) exon 3 ' terminal unpaired donor splicing site; Thereby generation transfectional cell;

(b) transfectional cell is remained under the condition that is suitable for homologous recombination, thereby produce the homologous recombination cell;

(c) the homologous recombination cell is remained under the condition that is suitable for expression of target gene under the promotor control.

88. the target gene expression by coded protein in the change cell prepares method of protein, comprises step:

(a) use the DNA construct transfectional cell, DNA construct comprises:

(i) with the coding region of target gene in or the genomic dna homologous targeting sequencing of upstream;

(ii) Actin muscle or collagen promotor; Thereby generation transfectional cell;

(b) transfectional cell is remained under the condition that is suitable for homologous recombination, thereby produce the homologous recombination cell;

(c) the homologous recombination cell is remained under the condition that is suitable for protein expression under the promotor control, wherein the target gene Actin muscle collagen of also not encoding of neither encoding.

89. one kind provides method of protein to Mammals, comprises any one cell of introducing claim 22-24 to mammalian cell, the wherein interior dna encoding the protein of giving birth to.

90. according to the method for claim 89, wherein cell before being incorporated into Mammals, seal up for safekeeping allow protein from barrier device inside in the barrier device that the barrier device outside is passed through.

91. according to the method for claim 90, wherein barrier device can prevent that cell from overflowing from barrier device.

92. according to the method for claim 89, wherein DNA construct further comprises the coding amplifiable marker, can choose the sequence of the cell that contains multiple copied coding amplifiable marker sequence; Before being incorporated into Mammals, cell is cultivated under the condition of choosing the cell that contains multiple copied coding amplifiable marker sequence.

93. according to the method for claim 92, wherein amplifiable marker is chosen from following group, comprises Tetrahydrofolate dehydrogenase, adenosine deaminase, trifunctional enzyme-carbamyl phosphate synthetase-aspartate transcarbamylase and dihydroorotase (CAD).

94. according to the method for claim 89, wherein cell is the human cell.

95. according to the method for claim 89, wherein cell is primary or secondary cell.

96. according to the method for claim 89, wherein cell is an immortalized cell.

97. according to the method for claim 89, wherein cell is the Mammals inoblast.

98. according to the method for claim 89, wherein cell is human fibroblast's a primary or secondary cell.

99. one kind prepares method of protein, comprising:

(a) provide HT1080 cell, contain and comprise and the sequence of the coded protein DNA of bonded external source regulation domain operationally,

(b) be suitable for preparing culturing cell under the proteinic condition, thereby producing protein,

(c) confirm to have produced protein,

Wherein, protein is chosen from following group, comprises alpha-interferon, beta-interferon, gamma-interferon, nerve growth factor, FSH β, TGF-β, tumour necrosis factor, hyperglycemic-glycogenolytic factor, bone growth factor-2, bone growth factor-7, TSH-β, interleukin 1, interleukin II, interleukin, interleukin 6, interleukin-11, interleukin 12, GSF-granulocyte (G-CSF), CSF-scavenger cell (M-CSF), CSF-granulocyte/scavenger cell (GM-CSF), protein kinase C, urokinase, Antithrombin III, DNA-se, alpha-galactosidase, tyrosine hydroxylase, blood coagulation factor V, blood coagulation factor VII, blood coagulation factor X, blood coagulation factor XI, plasma thromboplastin antecedent II, IL-2 antagonist and solvable CD4.

100. according to the method for claim 99, comprising:

(i) external source regulation domain;

The (ii) sequence of coded protein, DNA be incorporated in the precursor of HT1080 cell by transfection.

101. according to the method for claim 99, wherein protein is the blood coagulation Factor IX.

102. according to the method for claim 99, wherein protein is the blood coagulation factors IX.

103.DNA plasmid pREPO18.

104. according to the DNA construct of claim 25, target gene coding hemopoietin wherein.

105. according to the method for claim 41, target gene coding hemopoietin wherein.

106., give birth to the genes encoding hemopoietin in wherein according to the vertebrate cells of the cultivation of claim 60.

107. the plant of a cultivation or fungic origin cell, mix transcription unit in its genome, wherein transcription unit comprises external source adjusting sequence, the external source exon, with 3 ' of the external source exon terminal donor splicing site, all all are positioned at the upstream of the living transcription initiation site of giving birth to gene, and donor splicing site operationally combines with the second interior interior living acceptor splicing site of giving birth to exon of interior living gene.

108. one kind provides method of protein to Mammals, comprises to Mammals introducing claim 60-81 83,86 and 107 any one cell, the wherein interior dna encoding the protein of giving birth to.

109. according to the method for claim 108, wherein cell before being incorporated into Mammals, seal up for safekeeping allow protein from barrier device inside in the barrier device that the barrier device outside is passed through.

110. according to the method for claim 109, wherein barrier device can prevent that cell from overflowing from barrier device.

111. according to the method for claim 108, wherein DNA construct further comprises the coding amplifiable marker, can choose the sequence of the cell that contains multiple copied coding amplifiable marker sequence; Before being incorporated into Mammals, cell is cultivated under the condition of choosing the cell that contains multiple copied coding amplifiable marker sequence.

112. according to the method for claim 111, wherein amplifiable marker is chosen from following group, comprises Tetrahydrofolate dehydrogenase, adenosine deaminase, trifunctional enzyme-carbamyl phosphate synthetase-aspartate transcarbamylase and dihydroorotase (CAD).

113. according to the method for claim 108, wherein cell is the human cell.

114. according to the method for claim 108, wherein cell is primary or secondary cell.

115. according to the method for claim 108, wherein cell is an immortalized cell.

116. according to the method for claim 108, wherein cell is the Mammals inoblast.

117. according to the method for claim 108, wherein cell is human fibroblast's a primary or secondary cell.The claim 57-71 of the corresponding cancellation of claim 118-132.

118. a fusion rotein, by changing the method preparation of expression of target gene in the cell, method comprises step:

(a) provide cell, its genome comprises:

(i) contain interior target gene of giving birth to regulation domain;

(ii) target site;

(b) provide DNA construct, comprising:

(i) with target site homologous targeting sequencing;

(ii) external source is regulated sequence;

(iii) exon;

The amplifiable marker of (iv) encoding can be chosen and contain the multiple copied coding and can increase

The sequence of the cell of the sequence of mark;

(v) at exon 3 ' terminal unpaired donor splicing site,

(c) use the DNA construct transfectional cell, thereby produce transfectional cell;

(d) transfectional cell is remained under the condition that is suitable for homologous recombination, thereby produce the homologous recombination cell, except all exons of target gene, the genome of cell also comprises external source adjusting sequence, construct exon and the construct donor splicing site of deriving of deriving;

(e) the homologous recombination cell is cultivated under the condition of the cell of choosing the sequence that contains multiple copied coding amplifiable marker;

(f) the homologous recombination cell being remained on external source regulates under the condition that is suitable for transcribing under the sequence control, produce the construct exon of deriving, target gene, and be present in the derive transcription of any sequence between exon and the target gene of construct, wherein corresponding construct derive the transcription RNA of donor splicing site with corresponding to the directed montage of acceptor splicing site in the transcription in a site in the target gene;

(g) the homologous recombination cell is remained under the condition that is suitable for transcription montage and translation;

(h) confirmation has produced the translated product of transcription, wherein fusion rotein contains by derive first aminoacid sequence of exons coding and by second aminoacid sequence of part or all of target gene coding of construct, first aminoacid sequence be different from by target gene first in give birth to the aminoacid sequence of exons coding.

119. according to the fusion rotein of claim 118, wherein target gene is a hemopoietin.

120. according to the fusion rotein of claim 118, its sequence comprises the amino acid/11-3 of human growth hormone's signal peptide.

121. the vertebrate cells of a cultivation, mix transcription unit in its genome, wherein transcription unit comprises external source adjusting sequence, the external source exon, with 3 ' of the external source exon terminal donor splicing site, donor splicing site operationally combines with the second interior interior living acceptor splicing site of giving birth to exon of interior living gene, and wherein cell comprises construct deutero-amplifiable marker gene.

122. according to the vertebrate cells of the cultivation of claim 121, give birth to the protein that genes encoding is chosen in wherein from following group, comprising: regucalin, Regular Insulin, pancreotropic hormone, insulin-like growth factor, Rat parathyroid hormone 1-34, nerve growth factor, interleukin-, immunoglobulin (Ig), catalytic antibody, superoxide dismutase is organized the plasminogen activator, the blood coagulation Factor IX, apolipoprotein E, apolipoprotein A-I, globin, low density lipoprotein receptor, the IL-2 acceptor, the IL-2 receptor antagonist, α-1 antitrypsin, immune response modifier.

123., give birth to genes encoding tethelin in wherein according to the vertebrate cells of the cultivation of claim 121.

124., give birth to the genes encoding Interferon, rabbit in wherein according to the vertebrate cells of the cultivation of claim 121.

125., give birth to genes encoding blood coagulation factors IX in wherein according to the vertebrate cells of the cultivation of claim 121.

126. according to the vertebrate cells of the cultivation of claim 121, give birth to the genes encoding glucocerebrosidase in wherein,

127., give birth to the genes encoding G CFS in wherein according to the vertebrate cells of the cultivation of claim 121.

128., give birth to the genes encoding hemopoietin in wherein according to the vertebrate cells of the cultivation of claim 121.

129. the vertebrate cells of a cultivation, mix transcription unit in its genome, wherein transcription unit comprises external source adjusting sequence, the external source exon, with 3 ' of the external source exon terminal donor splicing site, all are positioned at the upstream of the living transcription initiation of giving birth to gene, and donor splicing site operationally combines with the second interior interior living acceptor splicing site of giving birth to exon of interior living gene, wherein cell is chosen from following group, comprise the Raji cell, Jurkat cell, Namalwa cell, the HL-60 cell, the Daudi cell, RPMI 8226 cells, U-937 cell, Bows melanoma cells, WI-38VA13 subspecies are cell and MOLT-4 cell.

130. one kind provides method of protein to Mammals, comprises the vertebrate cells of introducing any one cultivation of claim 121-129 to Mammals, the wherein interior dna encoding the protein of giving birth to.

131. according to the method for claim 130, wherein the vertebrate cells of Pei Yanging before being incorporated into Mammals, seal up for safekeeping allow protein from barrier device inside in the barrier device that the barrier device outside is passed through.

132. according to the method for claim 131, wherein barrier device can prevent that cell from overflowing from barrier device.

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