CN1748034B - Improved expression element - Google Patents

Abstract

The present invention relates to improved genetic elements that provide high levels of expression of operably linked genes in various tissues. In particular, unmethylated fragments, CpG islands of less than 2kb, are shown to provide enhanced transgene expression and have advantages in vector construction and cloning capacity.

Description

Improved expression element

field of invention

The present invention relates to polynucleotides comprising improved, smaller ubiquitous chromatin opening elements (UCOEs). When operably linked to an expressible nucleic acid sequence, the elements produce high and reproducible levels of gene expression. The present invention also relates to vectors containing said polynucleotide sequences, host cells containing said vectors and the use of said polynucleotides, vectors or host cells in therapy, or for expressing proteins in cell culture.

Background of the invention

Current models of chromatin structure in higher eukaryotes assume gene organization in a "domain format" (Dillon, N. & Grosveld, F. Chromatin Domains as Potential Eukaryotic Gene Functional Units, Curr. Opin. Genet. Dev .4, 260-264 (1994); Higgs, D.R. LCRs open chromatin domains? Cell 95, 299-302 (1998)). Chromatin domains are imagined to exist in a condensed, 'closed', transcriptionally silent state, or a decondensed, 'open', and transcriptionally active configuration. The establishment of an open chromatin structure, characterized by increased DNasel sensitivity, DNA hypermethylation and histone hyperacetylation, is thought to be necessary for initiating gene expression.

The nature of the opening and closing of chromatin regions is reflected in the behavior of transgenes randomly integrated into the host cell genome. The same construct produces different tissue-specific and developmental stage-specific expression patterns when integrated at different locations in the mouse genome (Palmiter, R.D. & Brinster, R.L. Ann. Ref. Genet. 20, 465-499 (1986); Allen , N.D.et al.Nature 333,852-855(1988); Bonnerot, C., Grimber, G., Briand, P. & Nicolas, J.F.Proc.Natl.Acad.Sci.USA 87:6331-6335(1990 )).

A variegated expression pattern is also frequently observed in a given transgenic mouse tissue, commonly referred to as position-effect variegated (PEV) (Kioussis, D. & Festenstein, R.Curr.Opin.Genet.Dev.7, 614- 619 (1997)). When exogenous genes are integrated into chromosomes of mammalian cells cultured in vitro, many integration events cause rapid silencing of transgenes, while others cause large changes in expression levels (Pikaart, M.J., Recillas-Targa, F. & Felsenfield, G . Genes Dev. 12, 2852-2862 (1998); Fussenegger M., Bailey, J. E., Hauser, H. & Mueller, P.P Trends Biotech. 17, 35-42 (1999)). These position effects reduce the efficiency of transgene expression, showing its application potential in basic research and biotechnology.

Chromatin domain models of gene organization suggest that genetic control elements that establish and maintain an open chromatin structure capable of transcriptional activity should be associated with active regions of the genome.

Locus control regions (LCRs) are a class of transcriptional regulatory elements capable of long-range chromatin remodeling. LCRs in transgenic mice are functionally defined by their ability to express expression at physiological levels, integration site-independent, transgene copy number-dependent, at cis-linked genes, especially single-copy transgenes, Fraser, P . & Grosveld, F. Curr. Opin. Cell Biol. 10, 361-365 (1998); Li, Q., Harju, S. & Peterson, K. R. Trends Genet. 15: 403-408 (1999). Importantly, this expression is tissue-specific. LCRs can hinder the dissemination of heterochromatin, prevent PEV (Kioussis, D. & Festenstein, R. Curr. Opin. Genet. Dev. 7, 614-619 (1997)), and by a series of genes located 5' to the genes they regulate or 3' DNase I hypersensitive (HS) site composition (Li, Q., Harju, S. & Peterson, K.R. Trends Genet. 15:403-408 (1999)).

LCRs appear to consist of two separate, though not necessarily independent, components. First, the establishment of an "open chromatin domain", and second, a pronounced translational activation capacity that results in transgene copy number-dependent expression (Fraser, P. & Grosveld, F. Curr. Opin. Cell Biol. 10, 361-365 (1998) The molecular mechanism by which LCRs exert their functions is still a matter of debate (Higgs, D.R. Cell 95, 299-302 (1998); Bulger, M. & Groudine, M. Genes Dev. 13, 2465-2477 (1999); Grosveld F. Curr. Opin. Genet Dev. 9152-157 (1999); Bender, M.A., Bulger, M., Close, J. & Groudine, M.. Mol. Cell 5, 387-393 (2000).

Proliferation of mammalian cell lines producing high levels of therapeutic protein products is a major developing industry. Chromatin position effects make this a difficult, time-consuming and costly process. The most commonly used method for producing such mammalian "cell factories" relies on gene amplification induced by combinations of drug resistance genes (e.g., DHFR, glutamine synthetase (Kaufman RJ. Methods in Enzymology 185, 537-566 (1990)), and maintain stringent selection pressure. By utilizing vectors containing LCRs from highly expressed gene domains, using cells from suitable tissues, the method is greatly simplified, producing a large proportion of clones showing high levels of expression Cell lines (Needham M, Gooding C, Hudson K, Antoniou M, Grosfeld F and Hollis M. Nucleic Acids Res 20, 997-1003 (1992); Needham M, Egerton M, Millest A, Evans S, Popplewell M, Cerillo G, McPheat J, Monk A, Jack A, Johnstone D, and Hollis M. Protein Expr Purif 6, 124-131 (1995).

However, the tissue specificity of LCRs, while useful in some circumstances, remains a controlling factor for many applications where expression is desired in tissues where no LCRs are known, or where expression is required in many or all tissues.

Our pending patent applications PCT/GB99/02357 (WO00/05393), US 09/358082, GB 0022995.5 and US 60/252,048, incorporated herein by reference, describe the processes responsible for the establishment of open chromatin in their natural chromosomal environment An element of a domain that spans a locus consisting only of ubiquitously expressed housekeeping genes. These elements are not from the LCR and include extended unmethylated CpG islands. We use the term ubiquitous chromatin opening element (UCOE) to describe this element.

In mammalian DNA, the dinucleotide CpG is identified by a DNA methyltransferase that methylates cytosine to 5-methylcytosine. However, 5-methylcytosine is unstable and is converted to thymine. As a result, CpG dinucleotides are generated much less frequently than by chance. However, some regions of genomic DNA have CpGs at a frequency close to that expected, and these sequences are called "CpG islands". As used herein, a "CpG island" is defined as a DNA sequence of at least 200 bp with a GC content of at least 50% and an observed/expected CpG content ratio of at least 0.6 (i.e., a CpG dinucleotide content of at least 60%) (Gardiner-Green M and Frommer M.J Mol Biol 196, 261-282 (1987); Rice P, Longden I and Bleasby A Trends Genet 16, 276-277 (2000).

Unmethylated CpG islands are well known in the art (Bird et al. (1985) Cell 40:91-99, Tazi and Bird (1990) Cell 60:909-920), and when a significant proportion of cytosine residues are unmethylated When ylated, they can be defined as CpG islands, and they usually extend beyond the 5' ends of two closely spaced (0.1-3 kb) divergently transcribed genes. These regions of DNA have been reported to remain hypermethylated throughout developmental stages in all tissues (Wise and Pravtcheva (1999) Genomics 60:258-271). They are often associated with the 5' ends of ubiquitously expressed genes, and an estimated 40% of genes show tissue-restricted expression profiles (Antequera, F. & Bird, A.Proc.Natl.Acad.Sci.USA 90, 1195-11999 (1993 ); Cross, S.H.&Bird, A.P.Curr.Opin, Genet.Dev.5, 309-314 (1995), and known to be the localization region of active chromatin (Tazi, J.&Bird, A.Cell, 60, 909- 920 (1990).

An 'extended' unmethylated CpG island is an unmethylated CpG island that spans a region comprising more than one transcription start site and/or spans more than 300 bp and preferably more than 500 bp. The boundaries of extended unmethylated CpG islands are determined by using PCR in this region in conjunction with the use of a restriction enzyme that has the ability to digest (cut) DNA at the recognition sequence and is sensitive to the methylation status of any CpG residues present. Limit the function. One such enzyme is Hpall, which recognizes and digests CCGG sites normally found within CpG islands, but only if the central CG residue is not methylated. Therefore, PCR performed with Hpall-digested DNA and the region containing the Hpall site, if the DNA is unmethylated, does not yield amplification products due to Hpall digestion. PCR will only produce amplification products if the DNA is methylated. Thus, PCR amplification products can be observed outside the unmethylated regions of Hpall-undigested DNA, thereby defining the boundaries of "extended unmethylated CpG islands".

We have demonstrated (WO 00/05393) that spanning DNA from human TATA-binding protein (TBP)/protein body component-B1 (PSMBI) and nuclear heterogeneous-ribonucleoprotein A2/B1 (hnRNPA2)/heterochromatin protein 1Hsy (HP1 ^Hsγ ) locus without methylated CpG island regions of the heteroduplex diverging transcriptional promoter, resulting in reproducible, physiological gene expression and the ability to prevent transgene integration that normally occurs within central heterochromatin Role of variegated expression patterns and silencing.

The term "reproducible expression" as used herein means that a polynucleotide of the invention expresses an expressible gene directly at substantially the same expression level regardless of its chromatin environment, preferably regardless of the cell type or organization type. Those skilled in the art will recognize that substantially the same level of expression of an operably linked expressible gene is obtained regardless of the claimed polynucleotide chromatin environment, and preferably regardless of the cell type, assuming that the cell is capable of actively expressing the gene.

We have shown (WO 00/05393) that unmethylated CpG islands associated with actively transcribed promoters have the ability to remodel chromatin and are thus considered to be fundamental determinants for the establishment and maintenance of open domains of housekeeping loci .

UCOEs increase the proportion of prolific gene delivery events by improving the level and stability of transgene expression. This has important research and biotechnology applications, including making transgenic animals and producing recombinant protein products in cultured cells. We have shown (WO 00/05393) the beneficial effect of UCOEs on the expression of a CMV-EGFP reporter gene construct and secreted pharmaceutically valuable erythropoietin protein. The nature of UCOEs also suggests utility in gene therapy, whose effectiveness is often limited by low frequency of prolific gene delivery and insufficient expression levels and duration (Verma, I.M. & Somia, N. Nature 389:239-242 (1997).

Our above-mentioned application PCT/GB99/02357 (WO 00/05393) discloses a functional UCOE fragment of about 4.0 kb, especially the '5.5RNP' fragment defined by nucleotides 4102 to 8286 of Figure 21 (disclosed in p11, lines 6 and 7). The same application discloses a '1.5kbRNP' fragment (Figures 22 and 29, source described in p51, lines 1 to 5). However, this fragment is actually a 2165bp BamH1-Tth111I fragment of the above '5.5RNP' fragment, consisting of nucleotides 4102 to 6267 of the application.

In a further application (WO 02/24930) we disclosed artificially constructed UCOEs comprising fragments of naturally occurring CpG islands. The disclosed fragments were larger than those claimed in the current application, and at the time it was not considered possible to use the small fragments alone, not just as components of synthetic or 'mixed' UCOE constructs.

Even with these striking implications and broad range of applications, there is still a need to further optimize transgene expression levels. There is a need to further optimize the level of transgene expression, especially in the field of gene therapy in vivo and in the field of recombinant protein production in vitro.

A particular need is to reduce the size of elements used to enhance gene expression. In this way, smaller vectors can be produced, or vectors that can stably accommodate and express larger inserts.

Brief description of the invention

It is an object of the present invention to provide small chromatin opening elements to enhance the level and reproducibility of operably linked transgenes.

According to the present invention, polynucleotides comprising small fragments of functional UCOEs are provided. Such polynucleotides include no more than about 2 kb unmethylated CpG islands, or fragments no more than about 2 kb larger than such islands.

Although large nucleotides comprising extended unmethylated CpG islands are known in the prior art (see applicant's own previous application WO 00/05393), it was not previously determined whether it was possible to use significantly smaller fragments and still maintain Enhancement of expression levels and consistency obtained from large UCOEs/unmethylated CpG islands.

The term "operably linked" as used herein refers to an operable relationship between polynucleotide elements of the invention. "Operably linked" is a term well known to those skilled in the art and describes a functional relationship between DNA sequences acting in cis. The exact structural relationships may or may not be associative, and differ for different element types. With respect to a promoter, it is implied to be located substantially adjacent (usually within less than 100 bp) to the 5' open reading frame it drives. In the case of extended unmethylated CpG islands, local effects on chromatin structure appear to be responsible for increasing the level and consistency of gene expression. For example, an element comprising an extended unmethylated CpG island is placed immediately 5' to a promoter that controls transcription of an expressible gene. However, "operably linked" embraces the possibility of it being placed elsewhere as long as a definite functional effect can be demonstrated.

The invention provides isolated polynucleotides comprising

a) Extended unmethylated CpG islands;

b) an expressible open reading frame operably linked to said extended unmethylated CpG island;

c) a promoter, operably linked to said open reading frame, wherein said promoter is not naturally linked to said CpG island;

It is characterized in that the size of said CpG island does not exceed 2 kb and wherein reproducible expression of said open reading frame is obtained in two tissue types.

Alternatively, the polynucleotide includes a fragment of no more than 2kb of the human hnRNP A2 gene, preferably no more than 1.6kb, including a 1546bp Esp31 restriction fragment, more preferably the sequence of Figure 2, 977 to 2522 nucleotides (SEQ ID NO: 1 ), or its functional analogues. Preferably, said fragments are oriented forward.

'Functional homologue' refers to a polynucleotide sequence capable of hybridizing to the disclosed sequence under stringent conditions and having a similar expressible open reading frame that is operably linked and repeatable in two or more tissues nature of expression. Stringent hybridization/wash conditions are known in the art. For example, stable nucleic acid hybrids after washing in 0.1x SSC, 0.1% SDS at 60°C. If the nucleic acid sequence is known, calculation of optimal hybridization conditions is well known in the art. For example, hybridization conditions can be determined according to the GC content of the nucleic acid to be hybridized. See Sambrook et al. (1989), Molecular Cloning; Laboratory Methods. A general formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a given similarity is:

T _m =81.5°C+16.6Log[Na+]+0.41[%G+C]-0.63(%formamide)

Preferably, the polynucleotides of the invention promote non-tissue-specific, reproducible expression of operably linked genes.

In another preferred scheme, the polynucleotide includes a fragment of no more than about 1 kb of the human hnRNP A2 gene, preferably includes a 987bp BspE1-Esp31 restriction fragment, more preferably includes the sequence of Figure 2, nucleotides from 1536 to 2522 (SEQ ID NO: 2).

Preferably, the polynucleotides of the invention include one or more naturally occurring promoters.

Preferably, the polynucleotides of the present invention include a dual or bidirectional promoter of divergent transcription. In other embodiments, the polynucleotides of the invention comprise fragments of the β-actin CpG island/promoter region, preferably of human origin. More preferably, the polynucleotide of the present invention comprises a DNA fragment spanning the CpG island/promoter region of human β-actin in the range of 100 bp to 2 kb.

In further optional embodiments, the polynucleotides of the invention comprise fragments of the PDCD2 CpG island/promoter region, preferably of human origin. More preferably, the polynucleotide of the present invention comprises a DNA fragment spanning the human PDCD2 CpG island/promoter region in the range of 100 bp to 2 kb.

Preferably, the polynucleotide of the present invention includes a DNA fragment spanning the CpG island/promoter region of human β-actin in the range of 100bp to 1.9kb and a DNA fragment spanning the CpG island/promoter region of human PDCD2 in the range of 100bp to 2kb. Preferably, said fragments are directly adjacent to their dc-oriented promoters.

In another aspect, the present invention provides a vector comprising the polynucleotide of the present invention disclosed above. Preferably, the vector is an expression vector adapted for eukaryotic gene expression.

Typically said adjustments include, for example but not limited to, provision of transcriptional control sequences (promoter sequences) that mediate cell/tissue specific expression.

Promoter and enhancer are art-recognized terms that include the following features, which are provided by way of example and not by way of limitation. A promoter is a 5', cis-acting regulatory sequence linked directly to the initiation of transcription. The promoter element includes the so-called TATA box and an RNA polymerase initiation selection (RIS) sequence for selecting where transcription starts. These sequences also bind polypeptides for RNA polymerase to facilitate transcription initiation selection.

Preferably, the promoter is selected from CMV, EF-la, Rous Sarcoma Virus (RSV) LTR, or HIV2 LTR or a combination sequence derived therefrom. More preferably, the promoter is the CMV immediate/early promoter. Most preferably it is the mouse CMV immediate/early promoter.

In the most preferred vector embodiments, the CpG islands of the invention are located adjacent to and 5' to an operable promoter controlling expression of an expressible open reading frame.

Enhancer elements are cis-acting nucleic acid sequences that are often found 5' to the transcription initiation site of a gene (enhancers can also be found 3' to a gene sequence or even in intronic sequences and are thus position independent). The role of an enhancer is to increase the rate of transcription of the gene linked to the enhancer. Enhancer activity corresponds to trans-acting transcription factors (polypeptides) that have been shown to bind specifically to enhancer elements. The binding/activity of transcription factors corresponds to a number of environmental factors including, for example, but not limited to, intermediate metabolites (eg, glucose), environmental effectors (eg, heat). (See Eukaryotic Transcription Factors, David S Latchman, Academic Press Ltd, SanDiego).

Modifications also include the provision of selectable markers and autonomously replicating sequences, both of which facilitate maintenance of the vector in eukaryotic or prokaryotic hosts. Self-sustaining vectors are called episomal vectors. Episomal vectors are desirable because they are self-replicating and persist without the need for integration. Such episomal vectors are described in WO98/07876.

Modifications of the vector to facilitate expression of the encoding gene include the provision of transcription termination/polyadenylation sequences. This also includes the provision of internal ribosome entry sites (IRES), which act to maximize expression of vector-encoded genes arranged in bi-cis or multi-cis expression cassettes.

These adjustments are known in the art. There is generally a large body of published literature on expression vector construction and recombinant DNA methods. See, Sambrook et al (1989) Molecular Cloning: A Laboratory Guide, Cold Spring Harbor, NY and references therein; Marston, F (1987) DNA Cloning: A Practical Approach Vol III IRL Press, Oxford UK; DNA Cloning: FM Ausubel et al., Current Handbook of Molecular Biology, John Wiley & Sons, Inc, (1994).

The vector may be an episomal vector or an integrative vector. Preferably, the vector is a plasmid. Alternatively, the vector may be a virus, such as an adenovirus, adeno-associated virus, herpes virus, vaccinia virus, lentivirus or other retrovirus.

Preferably the vector comprises an operably linked gene, which is a therapeutic nucleic acid. Such therapeutic nucleic acids may work by replacing or supplementing the function of defective genes that cause diseases such as cystic fibrosis, thalassemia, sickle cell anemia, Fanconi's anemia, hemophilia, severe combined immunodeficiency (SCID), phenylketonuria (PKU), alpha-1 antitrypsin deficiency, Duchenne muscle atrophy, ornithine transcarbamylase deficiency, or osteogenesis imperfecta. Alternatively, it may encode the selective expression of a cytotoxic agent or prodrug converting enzyme in target cells, such as malignant cancer cells, to kill said cells. This use, and many others, are well known to those skilled in the art, and the relevance of the invention to enhance expression of therapeutic nucleic acids is clear to said skilled artisan.

Most preferably, said vector comprises any one of SEQ ID Nos 1 or 2, CMV promoter, multiple cloning site, polyadenylation sequence and a selectable marker encoding a suitable control element.

Host cells transfected with the vector are also provided. Preferably they are eukaryotic cells, more preferably mammalian cells, most preferably human or rodent cells.

Or, maybe plant cells.

A further aspect of the invention is any isolated polynucleotide, CpG island without methylation extension (in particular disclosed in SEQ ID Nos 1 or 2), vector or host cell disclosed in the above-mentioned treatment in particular in gene therapy in the application.

The present invention also provides the application of polynucleotide, vector or host cell in the preparation of medicine or composition for gene therapy.

The present invention also provides methods of treatment comprising administering an effective dose of said polynucleotides, vectors or host cells of the present invention to a patient in need of such treatment. Preferably the patient suffers from a disease amenable to gene therapy.

The present invention also provides pharmaceutical compositions, which include polynucleotides and/or vectors and/or host cells, optionally mixed with pharmaceutically acceptable carriers or diluents for treating diseases or providing specific tissues with beneficial proteins or functions Cell.

The polynucleotides, vectors or host cells or pharmaceutical compositions of the invention may be administered by routes including systemic intramuscular, intravenous, aerosol, oral (solid or liquid), topical, ocular, rectal, peritoneal Intra and/or intrathecal and local direct injection.

The exact dosage regimen will, of course, need to be determined by the individual clinician for the individual patient, which, in turn, will be governed by the desired gene expressed protein and the exact nature of the tissue type being targeted for treatment.

Dosage also depends on disease indications and route of administration. The number of doses depends on the disease, and efficacy data from clinical trials.

The amount of polynucleotide or vector DNA delivered according to the present invention for effective gene therapy is preferably in the range of 50 ng-1000 μg vector DNA/kg body weight; more preferably in the range of about 1-100 μg vector DNA/kg.

Although administration of polynucleotides, vectors or host cells to mammals for in vivo cellular uptake is preferred according to the invention, ex vivo methods can be utilized whereby cells are removed from the animal, transduced with the polynucleotide or vector, and then replanted into the animal. The liver, for example, can be used ex vivo by removing the hepatocytes from the animal, transducing the hepatocytes in vitro and reimplanting the transduced hepatocytes into the animal (as described in Chowdhury et al., Science 254:1802-1805, 1991 for rabbits, or Wilson, Hμm. Gene Ther. 3: 179-222, 1992 described for humans) for evaluation. This approach can also efficiently deliver different cell populations in the circulatory or lymphatic system, such as red blood cells, T cells, B cells, and hematopoietic stem cells.

In a further aspect, the present invention provides the use of the polynucleotide vector or host cell of the present invention in a cell culture system to obtain a desired gene product. Preferably, the expressible nucleic acid encodes a recombinant protein for expression in an in vitro cell culture system.

Suitable cell culture systems are known in the art and are fully described in the literature known to those skilled in the art. The invention provides a method for producing a polypeptide of the invention, comprising:

i) providing a cell transformed/transfected with a polynucleotide of the present invention;

ii) culturing said cell under conditions conducive to production of said polypeptide; and

iii) purifying said polypeptide from said cell or its growth environment.

Alternatively, the expressible gene encodes a non-polypeptide product, such as RNA. Such RNA may be antisense RNA capable of inhibiting expression of a specific gene at the post-translational level, or may be enzymatic (ribozyme) or otherwise functional, such as that of ribosomal RNA.

Also provided according to SEQ ID NOs: 1 or 2 extended non-methylated CpG island polynucleotides for improving the application of endogenous gene expression, comprising the polynucleotide inserted into the genome of the cell and endogenous The position of the source gene can be operably linked to increase the expression level of the gene.

In another embodiment of the invention there is provided a non-human transgenic animal comprising any polynucleotide or vector of the invention or any extended unmethylated CpG island of the invention, wherein said CpG island has been introduced manually. Production of transgenic mice (Gordon et al., Proc.Natl.Acad.Sci.USA 77:7380 (1980); Harbers et al., Nature 293:540 (1981); Wagner et al., Proc.Natl.Acad.Sci.USA 78:5016 (1981); and Wagner et al., Proc.Natl.Acad.Sci.USA 78:6376 (1981), sheep, pig, chicken (seeing Hammer et al., Nature 315:680 (1985)) etc. are prior art known and contemplated for use in the present invention.

Such transgenic animals containing the polynucleotide of the present invention can also be used for long-term production of desired proteins.

The invention also provides mammalian models for determining the efficacy of gene therapy using the polynucleotides, vectors or host cells of the invention. Mammalian models include transgenic animals whose cells contain a vector of the invention. The animals are available for testing before human clinical trials.

The present invention also provides the use of polynucleotides and extended unmethylated CpG islands of the present invention in the production of transgenic plants.

The production of transgenic plants with increased yield, or increased resistance to disease, pests, drought or salt is known to those skilled in the art. The invention also provides a transgenic plant comprising a cell comprising a polynucleotide of the invention. Some or all of the cells comprising a polynucleotide of the invention may be derived from plants.

The present invention also relates to the use of polynucleotides, vectors or extended unmethylated CpG islands of the present invention in functional genomics. Functional genomics is primarily concerned with the identification of genes specifically expressed in specific cell types or disease states, and currently provides thousands of new gene sequences that are of potential value for drug discovery or gene therapy purposes. A major problem in using this information to develop new treatments is how to determine the function of these genes. The polypeptides of the invention can be used in a variety of functional genomics applications to determine the function of a gene sequence. Functional genomics applications of the present invention include, but are not limited to:

(1) Using the polynucleotide of the present invention to obtain the continuous expression of the antisense sequence of the gene sequence or ribozyme knockdown library, so as to determine the effect of gene inactivation on the cell phenotype.

(2) Using the polynucleotide of the present invention to prepare an expression library of gene sequences, so that the gene sequences can be delivered into cells to produce reliable, reproducible and sustained expression of the gene sequences. The resulting cells expressing the gene sequence can be used in various functional identification and drug discovery methods. For example, to prepare neutralizing antibodies to gene products; to rapidly purify the protein product of the gene itself for structural, functional or drug screening studies; or for cell-based drug screening.

(3) In a method of using the polynucleotide of the present invention for mouse embryonic stem (ES) cells and transgenic mice. One of the most powerful functional genomic approaches involves the random insertion into the genes of mouse ES cells of constructs that allow drug selection only after insertion into the expressed gene and which can be easily cloned for sequencing (G. Hicks et al., Nature Genetics, 16, 338-334). Transgenic mice with knockout mutations and new sequences can thus be readily used to study their function. Currently the technique is useful for the 10% of mouse genes that are well expressed in mouse ES cells. Incorporation of polynucleotides of the invention into integrated constructs will extend these methods to identify all genes expressed in mice.

Detailed description of the invention

The present invention is only described by way of example with reference to the following drawings, wherein;

Figure 1 shows a map of the HP-1/hnRNPA2 locus showing 4, 2, 1.5 and 1 kb CpG island fragments that define differently for BamH1, HindIII, Esp31, Tth1111 and BspEI restriction sites.

Figure 2 shows the nucleotide sequence of the HP-1/hnRNPA2 locus indicating BamH1, Esp31, BspE1 and Tth111I restriction sites.

Figure 3 shows the expression of an EGFP reporter gene operably linked to 4 kb and 1.5 kb (in both orientations) CpG island fragments. FACScan data are expressed as median fluorescence over 71 days.

Figure 4 shows the expression of an EGFP reporter gene operably linked to 4 kb and 1.5 kb (in both orientations) CpG island fragments. FACScan data are expressed as % positive cells over 71 days.

Figure 5 shows the expression of an EGFP reporter gene operably linked to 4 kb, 1.5 kb and 1 kb CpG island fragments. FACScan data are expressed as median fluorescence over 68 days.

Figure 6 shows the expression of an EGFP reporter gene operably linked to 4 kb, 1.5 kb and 1 kb CpG island fragments. FACScan data are expressed as % positive cells over 68 days.

Figure 7 shows the structure of two adenovirus constructs, Ad.CMV-Luc-SV40p (A) and Ad.1.5kb(F)UCOE-CMV-Luc-SV40p (A), which are based on adenovirus serotype 5. The luciferase expression cassette was inserted in the El region in a left-to-right orientation. The E1 and E3 regions are deleted from the virus. Due to the deletion of El, the virus is replication deficient. CMV (cytomegalovirus) is the human CMV enhancer/promoter. SV40p (A) is the late polyadenylation signal of SV40 virus from pGL3basic (Promega).

Figure 8 shows that 1.5kb UCOE increases gene expression in HeLa cells when delivered by an adenoviral vector. HeLa cells were infected with Ad.CMV-Luc-SV40p (A) and Ad.1.5kb (F) UCOE-CMV-Luc-SV40p (A) virus in 200μl infection medium at MOI50 for 2-3 hours (normal HeLa medium only contained 1% FCS). 2 ml of complete medium was added after incubation, and cells were seeded in 6-well plates. Luciferase activity was analyzed 2 days after infection. Results of three independent experiments are shown. Experiments were performed in triplicate. Displayed as mean +/- standard deviation.

Figure 9 shows that the 1.5kb UCOE effect is independent of virus preparation. HeLa cells were treated with viruses Ad.CMV-Luc-SV40p (A) and Ad.1.5kb (F) UCOE- CMV-Luc-SV40p (A) were infected for 2-3 hours respectively. After incubation 2ml of complete medium was added and cells were seeded in 6-well plates. Luciferase activity was analyzed 2 days after infection. Shows the results of a typical experiment. Experiments were performed in triplicate.

Figure 10 shows that 1.5kb UCOE increases transgene expression levels and stability in retrovirally transduced CHO-K1 aggregates. (A) Mean GFP values versus time for FACS-sorted CHO-K1 pools (36,000 cells initially FACS-sorted) following retroviral transduction of CMV and 1.5 UCOE-CMV constructs. Day 1 refers to the first day of measurement (8 days after FACS sorting). (B) Histogram of FACS-sorted CHO-K1 aggregates at each measurement time point.

Figure 11 shows GFP expression of transduced CHO-K1 (pool I) after low MOI infection. (A) Mean GFP values of the GFP-positive (M1) CHO-K1 population at various time points after retroviral transduction. The CMV population started with 3.19% GFP positive cells and the 1.5UCOE-CMV population started with 4.99% GFP positive cells. (B) Histogram of cell populations at various time points.

Figure 12 shows GFP expression of transduced CHO-K11 (pool II) after medium MOI infection. (A) Mean GFP values of the GFP-positive (M1) CHO-K1 population at various time points after retroviral transduction. The CMV population started with 25.2% GFP positive cells and the 1.5UCOE-CMV population started with 14.35% GFP positive cells. (B) Histogram of cell populations at various time points.

Figure 13 shows that 1.5 kb UCOE increases transgene expression levels and stability in retrovirally transduced HeLa aggregates. (A) Mean GFP values versus time for FACS-sorted HeLa pools (10,000 cells initially FACS-sorted) after retroviral transduction of CMV and 1.5 UCOE-CMV constructs. Day 1 refers to the first day of measurement (5 days after FACS sorting). (B) Histogram of FACS-sorted HeLa aggregates at each measurement time point.

Figure 14 shows that 1.5 kb UCOE increases the consistency of GFP expression in and among retrovirally transduced HeLa cell clones. FACS sorted HeLa clones were expanded to a 6-well size and analyzed for GFP expression. (A-C) Histograms of all CMV clones. (D-E) Histograms of all 1.5 UCOE-CMV clones.

Figure 15 shows that 1.5 kb UCOE reduces the coefficient of variation (CV) of GFP expression in HeLa cell clones. The mean CV values (+/- SD) of the mean of all retrovirally transduced and FACS sorted HeLa clones of Figure 5 are shown. The CV and its standard deviation (SD) of the 1.5 UCOE-CMV clone were significantly lower compared to the CMV clone.

Example

Example 1A 1.5kb HP-1/hnRNP A2UCOE enhanced expression

Materials and methods

Vector construction

As described in our prior application WO 00/05393, vector CET20 was obtained by cloning the 8.3 kb HindIII fragment of the human HP-1/hnRNP A2 locus (which contains the HP1/RNP promoter and extended CpG island) into generated in pBluescript (Stratagene).

The inserted 4186bp (referred to as 4kb) fragment was then removed by BamHI and HindIII digestion. These fragments were blunt-ended using T4 DNA polymerase and ligated into pEGFPN-1 (Clontech) which had been digested with Asel and blunt-ended again using T4 DNA polymerase. Clones with fragments in both orientations were then isolated.

A 1546bp Esp31 (isoschizase of BsmBI) fragment (referred to as a 1.5kb fragment) was again isolated from CET20 by Esp31(BsmBI) digestion followed by blunt-end filling, and these were then ligated into Asel of pEGFPN-1 as described above locus, clones that identify fragments in both orientations ("forward" orientation with the RNPA2 promoter having the same 5' to 3' orientation as the adjacent downstream operating CMV promoter that transcribes the EGFP transgene.)

transfection

Transfection of CHO-K1 cells according to standard methods was selected in G418, as described in the co-pending application incorporated herein by reference.

GFP expression analysis

Transfected cells were cultured in 600 μg/ml G418 selection medium. Cells were washed with standard phosphate buffered saline and stripped from the matrix with trypsin/EDTA according to standard protocols. Excess nutrient mixture F12 (HAM) medium (Gibco) was added and cells were transferred to 5 ml round bottom polystyrene tubes for Becton Dickinson FACScan analysis. GFP fluorescence was detected and compared to the autofluorescence of the parental cell population. Expression in cell populations can be expressed either as median fluorescence of an arbitrary group of units scaled linearly in the graph relative to controls (according to standard methods), or as % of cells identified as positive expressors.

result

As shown in Figure 3, from the perspective of fluorescence median, the expression was significantly enhanced (about 10 times, lasting at least 70 days) when the 1.5 kb fragment was inserted in the forward direction compared with the control, and an increase of about 60% was observed for the 4 kb fragment. In the experiments shown in Figure 5, the expression from the forward 1.5 kb fragment was comparable to that obtained with the 4 kb fragment. However, the opposite direction appears to be less effective from the perspective of the median fluorescence, as shown in Figure 3.

Figure 4 shows that both directions are comparable in terms of % positive cells. However, these data show that fragments may have some degree of directionality and that, for most cases, forward orientation is preferred.

Embodiment 2A 1kb HP-1/hnRNP A2UCOE enhanced expression

Materials and methods

Vector construction

The vector containing 1kb UCOE was digested with PciI and BspEI to remove the 5'500bp of the pEGFPN-1 vector with the 1.5kbEsp31 fragment in the forward direction, which was filled in and reconnected. This produced a vector with the 987bp BspE1-Esp31 fragment in only one orientation.

result

The forward 1 kb (987 bp) fragment showed comparable fluorescence median (Figure 5) and positive cell % (Figure 6) to the forward 1.5 kb fragment

Example 3A 1.5kb HP-1/hnRNP A2UCOE enhances adenovirus-encoded constructs Express

Materials and methods

cell culture

HeLa was obtained from ATCC (Manassas, Virginia). PER.C6 was obtained from Crucell, (Leiden, The Netherlands). All purchased cell lines were cultured as recommended by the manufacturer. 911 cells were kindly provided by Prof. L.S. Young (Cancer Research UK Institute for Cancer Studies, University of Birmingham, Birmingham, UK) and cultured in DMEM/10% FCS containing antibiotics.

Plasmid construction

PGL3basic was obtained from Promega (Madison, WI, USA) and contained the luciferase-SV40p (A) cassette downstream of the multiple cloning site. The human CMV enhancer/promoter (0.9 kb) was cloned into Smal digested pGL3basic to make pGL3/CMV-Luc-SV40p (A). To make pGL3/1.5kb(F)UCOE-CMV-SV40p(A), the 1.5kb UCOE Esp3I fragment (see Figures 1 and 2) was blunt-ended with T4 DNA polymerase (NEB, Beverly, MA, USA) and ligated into Nhel digested and T4 blunt-ended pGL3basic/CMV-Luc.

Viral vector construction

To construct pPS1128/CMV-Luc-SV40p(A) and pPS1128/1.5kb(F)UCOE-CMV-Luc-SV40p(A), the expression cassette was digested from pGL3/CMV-Luc-SV40p( Excised in A) and digested by KpnI/BamHI from pGL3/1.5kb(F)UCOE-CMV-Luc-SV40p (A), both expression cassettes were then blunt-ended and cloned into SpeI-digested blunt-ended in pPS1128 (Djeha et al., Cancer Gene Therapy 2000:721-731). Plasmids were analyzed by restriction enzyme digestion.

Viruses Ad.CMV-Luc-SV40p (A) and Ad.1.5kb (F) UCOE-CMV-Luc-SV40p (A) were obtained by using pPS1128/CMV-Luc-SV40p (A) and pPS1128/ 1.5kb (F) UCOE-CMV-Luc-SV40p (A) and overlapping adenovirus backbone vector pPS1160 were constructed by homologous recombination (Djeha et al., Cancer Gene Therapy 2000: 721-731), amplification and CsCI purification and titer determination as Described elsewhere (Lipinski et al., Gene Therapy, 8, 2001:274-281).

Virus infection and luciferase activity assay

HeLa cells (5.0×10 ⁴ per 6 wells) were infected with 200 μl infection medium suspension (EMEM/1% FCS with antibiotics) containing the corresponding virus for 2-3 hours. Then 1 ml of complete medium was added, and the cells were seeded into 6-well plates. Whole cell extracts were prepared by lysing cells in 200 μl lysis buffer (10 mM sodium phosphate pH 7.8, 8 mM MgCl ₂ , 1 mM EDTA, 1% Triton-X-100, 15% glycerol) and centrifuged (1 min, 13,000× g, RT) to obtain a clear lysate. The luciferase activity of the individual supernatants was analyzed at the indicated time points using a luminometer (Lumat LB 9501, Berthold, Wildbad, Germany) in the linear range.

result

To analyze whether UCOE can enhance gene expression in adenoviral vectors, 1.5kb UCOE was cloned in forward orientation upstream of the human CMV enhancer/promoter driving the luciferase reporter gene (RNP promoter has the same orientation as the CMV promoter) . Figure 7 schematically shows the structures of two viruses comparing their luciferase activities. HeLa cells were infected with the corresponding viruses at MOI 50, and the luciferase activity was analyzed 2 days after infection. Figure 8 clearly shows that the 1.5 kb UCOE fragment can greatly increase the expression level of the reporter gene in HeLa cells. To rule out specific effects related to virus preparations, we prepared new preparations of two viruses and repeated the experiment. Figure 9 shows that the UCOE effect is independent of the virus formulation and is fully reproducible with the new independent formulation.

Embodiment 4 A1.5kb HP-1/hnRNP A2UCOE enhances the construct encoded by retrovirus expression in

Materials and methods

cell culture

HeLa, CHO-K1 and 293 were obtained from ATCC (Manassas, Virginia). Both HeLa and 293 were cultured in DMEM supplemented with 1% NEAA and 10% FCS with antibiotics. CHO-K1 was cultured in nutrient mixed F12 (HAM) nutrient medium containing 10% FCS and antibiotics.

Plasmid construction

Plasmids pVPack-GP and pVPack-VSV-G were obtained from Stratagene (LaJolla, CA, USA) and express retroviral gag-pol and envelope VSV-G proteins, respectively. The retroviral vector pQCXIX was obtained from BD Bioscience, which contains the human CMV promoter for transgene expression (Palo Alto, CA, USA). Plasmid phrGFP-1 was obtained from Stratagene and used as the source of modified GFP cDNA. To clone pQCXIX-CMV-hrGFP, the hrGFP cDNA was excised from phr-GFP-1 by BamHI/EcoRV digestion and cloned into BamHI/EcoRV digested pQCXIX. To generate pQCXIX-1.5UCOE-CMV-hrGFP, the 1.5 kb UCOE fragment (Esp3I/BsmBI fragment) was cloned as a blunt-ended fragment into XbaI digested and blunt-ended pQCXIX-CMV-hrGFP.

Preparation of amphotropic VSV-G-enveloped retroviral particles

All viruses were pseudovirused with the amphotropic VSV-G (vesicular stomatitis virus glycoprotein), which produces efficient and broad host range transduction. The retroviral vector used produces a self-inactivating virus due to deletion of the U3 region of the 3'LTR copied to the 5'LTR after reverse transcription thus inhibiting any further transcription from the 5'LTR.

4.5 x 10 ⁶ 293 cells (cells should be 80-90% confluent the next day) were seeded in 8.4 cm diameter (55.6 cm ² ) dishes coated with type I collagenase the day before transient transfection. The next day 114 μl Lipofectamine 2000 (Invitrogen, Groenningen, The Netherlands) was mixed with 286 μl OptiMEM (Invitrogen) and incubated at room temperature (RT) for 5 minutes. In parallel, 1.5 μg of each of the plasmids pVPack-GP, pVPack-VSV-G and retroviral vectors were mixed with OptiMEM to achieve a final volume of 400 μl. The DNA mixture was added to the Lipofectamine 2000 solution and incubated at RT for 20-30 minutes. During the incubation period 293 cells were washed once with PBS and 6 ml of OptiMEM/10% FCS was added to the cells. Finally, the DNA/Lipofectamine mixture was added dropwise to the cells, and the cells were incubated at 37°C for 5 hours in a 5% CO ₂ incubator. The medium was then replaced with 8 ml of fresh 293 medium, and the cells were further cultured for 24-36 hours. Finally, the virion-containing supernatant was harvested from the cells, centrifuged at 1000 RPM for 5 minutes to pellet cells and cell debris, and the supernatant was filter-sterilized using a Millex-HV PVDF low protein binding 0.45 μm filter (Millipore, Molsheim, France) . The supernatant was aliquoted, snap frozen in liquid nitrogen and stored at -80°C.

Retroviral transduction of target cell lines

Target cells (HeLa, CHO-K1) were seeded into 6-well plates at 1×10 ⁵ cells/well the day before infection. For viral transduction, various amounts of 1,5-dimethyl-1,5-diazaundecylidene polymethyl bromide (Sigma, St. Louis, MO, USA) were added in the presence of 8.0 μg/ml virus-containing supernatant. Cells were incubated with virus for 24 hours, and then the medium was replaced with fresh medium. Gene expression can be observed two days after transduction. To prevent multiple copy effects per cell, target cells were infected in all assays with an amount of virus that yielded much less than 100% transduction efficiency (typically given 1-20% GFP positive cells). Within this range of transduction efficiencies, the volume of supernatant used for infection correlated linearly with the number of positive cells while mean expression levels showed a much lower increase.

FACS analysis

For FACS analysis, target cells were washed with PBS, trypsinized and resuspended in complete medium with a Becton Dickinson FACScan (BD, Franklin Lakes, NJ, USA) using constant instrument settings for each analysis Analysis of hrGFP (green fluorescent protein) expression. Data were analyzed with CeIIQuest software for Apple Mcintosh.

FACS sorting

To sort GFP-expressing HeLa and CHO-K1 cells, cells were prepared for FACS analysis and then sorted with a BD Bioscience FACS sorter (gift from The Institute for Cancer Studies, University of Birmingham, UK). Sorted individual clones and pools are then expanded and then express GFP over a period of time.

result

In the first study, CHO-K1 cells were transduced with supernatants containing retrovirus CMV-hrGFP (CMV) and 1.5UCOE CMV-hrGFP (1.5UCOE-CMV) cells. CHO-K1 cells were transduced as described in Materials and Methods, and three days after infection GFP-positive cells were FACS sorted into population pools (36,000 cells per cell construct) and expanded. GFP expression and histogram plots were performed over time and are illustrated in Figures 10A and B, respectively. On day 17 of the analysis, the mean of 1.5 UCOE-CMV aggregates was 2.6-fold higher than the mean of CMV aggregates. In addition, as shown in the histogram of Figure 10B, the 1.5 UCOE-CMV aggregate produced a denser GFP expression peak than the CMV aggregate. The average CV value (coefficient of variation; this is a marker for the uniformity of GFP expression) of GFP-positive cells was 146.0 for the CMV pool in Marker 1 and 83.0 for the 1.5 UCOE-CMV pool.

In contrast to this study, where CHO-K1 was transduced, the cell population was studied over time without FACS sorting. An MOI yielding a low percentage of positive cells was chosen to rule out multi-copy effects per cell. As shown in FIG. 11A (pool I with approximately 5% GFP positive cells), the mean value of positive cells in the 1.5 UCOE-CMV population at the last measurement time point was 2.8 times higher than the mean value of the CMV population. For pool II (approximately 15-25% GFP positive cells; FIG. 12A ), the mean value of positive cells for the 1.5 UCOE-CMV population at the latest measurement time point was 2.3-fold higher than the mean value of the CMV population.

Compared to the FACS sorted population, the 1.5UCOE-CMV population produced significantly denser expression peaks compared to the CMV population at all time points analyzed (Figure 1 IB and Figure 12B).

As for CHO-K1, pools of 10,000 HeLa cells stably transduced with the retroviral construct were subjected to FACS sorting. In Figure 13A, mean GFP values are shown as mean including the day of FACS sorting (day 5). At the last measurement, the average value of 1.5 UCOE-CMV aggregates was 1.6 times higher than the average value of CMV aggregates. As shown in the histogram of Figure 13B, many CMV cells lost their high GFP expression levels over time; in contrast, most of the 1.5UCOE-CMV cells maintained their high GFP expression.

HeLa single cell clones were also FACS-picked and expanded into 6-well plates. CMV clones tended to produce slightly higher expression peaks than 1.5 UCOE-CMV clones, however, both within and between clones, as shown in the histograms of all clones analyzed (Figure 14A-E), due to the presence of UCOE, Consistency of expression levels was significantly increased. Figure 15 shows the mean coefficient (CV) of GFP expression difference and its standard difference (SD). The CV+/-SD value of the CMV clone was 135.0+/-165.3 and that of the 1.5UCOE-CMV clone was 49.4+/-10.9.

sequence listing

<110>ML Laboratories PLC

<120> Improved expression elements

<130>P101405

<160>2

<170>PatentIn version 3.1

<210>1

<211>1546

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<213>Homo sapiens

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the

ggccctccgc gcctacagct caagccacat ccgaaggggg agggagccgg gagctgcgcg 60

cggggccgcc ggggggaggg gtggcaccgc ccacgccggg cggccacgaa gggcggggca 120

gcgggcgcgc gcgcggcggg gggaggggcc ggcgccgcgc ccgctgggaa ttggggccct 180

aggggggaggg cggaggcgcc gacgaccgcg gcacttaccg ttcgcggcgt ggcgcccggt 240

ggtccccaag gggagggaag ggggaggcgg ggcgaggaca gtgaccggag tctcctcagc 300

ggtggctttt ctgcttggca gcctcagcgg ctggcgccaa aaccggactc cgcccacttc 360

ctcgcccgcc ggtgcgaggg tgtggaatcc tccagacgct gggggagggg gagttggggag 420

cttaaaaact agtacccctt tgggaccact ttcagcagcg aactctcctg tacaccaggg 480

gtcagttcca cagacgcggg ccagggggtgg gtcattgcgg cgtgaacaat aatttgacta 540

gaagttgatt cgggtgtttc cggaaggggc cgagtcaatc cgccgagttg gggcacggaa 600

aacaaaaagg gaaggctact aagatttttc tggcgggggt tatcattggc gtaactgcag 660

ggaccacctc ccgggttgag ggggctggat ctccaggctg cggattaagc ccctcccgtc 720

ggcgttaatt tcaaactgcg cgacgtttct cacctgcctt cgccaaggca ggggccggga 780

ccctattcca agaggtagta actagcagga ctctagcctt ccgcaattca ttgagcgcat 840

ttacggaagt aacgtcgggt actgtctctg gccgcaaggg tgggaggagt acgcatttgg 900

cgtaaggtgg ggcgtagagc cttcccgcca ttggcggcgg atagggcgtt tacgcgacgg 960

cctgacgtag cggaagacgc gttagtgggg gggaaggttc tagaaaagcg gcggcagcgg 1020

ctctagcggc agtagcagca gcgccgggtc ccgtgcggag gtgctcctcg cagagttgtt 1080

tctcgagcag cggcagttct cactacagcg ccaggacgag tccggttcgt gttcgtccgc 1140

ggagatctct ctcatctcgc tcggctgcgg gaaatcgggc tgaagcgact gagtccgcga 1200

tggaggtaac gggtttgaaa tcaatgagtt attgaaaagg gcatggcgag gccgttggcg 1260

cctcagtgga agtcggccag ccgcctccgt gggagagagg caggaaatcg gaccaattca 1320

gtagcagtgg ggcttaaggt ttatgaacgg ggtcttgagc ggaggcctga gcgtacaaac 1380

agcttcccca ccctcagcct cccggcgcca tttcccttca ctgggggtgg gggatgggga 1440

gctttcacat ggcggacgct gccccgctgg ggtgaaagtg gggcgcggag gcgggaattc 1500

ttatccctt tctaaagcac gctgcttcgg gggccacggc gtctcc 1546

the

<210>2

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<213>Homo sapiens

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the

ccggaagggg ccgagtcaat ccgccgagtt ggggcacgga aaacaaaaag ggaaggctac 60

taagattttt ctggcggggg ttatcattgg cgtaactgca gggaccacct cccgggttga 120

gggggctgga tctccaggct gcggattaag cccctcccgt cggcgttaat ttcaaactgc 180

gcgacgtttc tcacctgcct tcgccaaggc aggggccggg accctattcc aagaggtagt 240

aactagcagg actctagcct tccgcaattc attgagcgca tttacggaag taacgtcggg 300

tactgtctct ggccgcaagg gtgggaggag tacgcatttg gcgtaaggtg gggcgtagag 360

ccttcccgcc attggcggcg gatagggcgt ttacgcgacg gcctgacgta gcggaagacg 420

cgttagtggg ggggaaggtt ctagaaaagc ggcggcagcg gctctagcgg cagtagcagc 480

agcgccgggt cccgtgcgga ggtgctcctc gcagagttgt ttctcgagca gcggcagttc 540

tcactacagc gccaggacga gtccggttcg tgttcgtccg cggagatctc tctcatctcg 600

ctcggctgcg ggaaatcggg ctgaagcgac tgagtccgcg atggaggtaa cgggtttgaa 660

atcaatgagt tattgaaaag ggcatggcga ggccgttggc gcctcagtgg aagtcggcca 720

gccgcctccg tgggagagag gcaggaaatc ggaccaattc agtagcagtg gggcttaagg 780

tttatgaacg gggtcttgag cggaggcctg agcgtacaaa cagcttcccc accctcagcc 840

tcccggcgcc atttcccttc actgggggtg ggggatgggg agctttcaca tggcggacgc 900

tgccccgctg gggtgaaagt ggggcgcgga ggcgggaatt cttattccct ttctaaagca 960

cgctgcttcg ggggccacgg cgtctcc 987

Claims (20)

1. isolating polynucleotide comprise

A) the no methylated CpG island of Yan Shening;

B) effable open reading frame can be operationally connected to the no methylated CpG island of described extension;

C) promotor can be operationally connected to described open reading frame, and wherein said promotor is not to be connected to described CpG island natively;

It is characterized in that described CpG island comprises the fragment of the people HP-1/hnRNPA2 locus that is no more than 1.6kb, and the repeating to be expressed in the two or more types of organizations and obtain of wherein said open reading frame.

2. the polynucleotide of claim 1 comprise the Esp31 restriction fragment.

3. the polynucleotide of claim 2 comprise the sequence of Nucleotide 977 to 2522 among the Fig. 2 shown in the SEQ ID NO:1.

4. each polynucleotide of claim 1 to 3 comprise the fragment of the HP-1/hnRNPA2 locus that is no more than 1kb.

5. the polynucleotide of claim 4 comprise the BspE1-Esp31 restriction fragment.

6. the polynucleotide of claim 5 comprise the sequence of Nucleotide 1536 to 2522 among the Fig. 2 shown in the SEQ ID NO:2.

7. carrier contains right and requires 1 to 6 each polynucleotide.

8. the carrier of claim 7, carrier wherein is an episomal vector.

9. the carrier of claim 7, carrier wherein is an integrating vector.

10. each carrier of claim 7-9, carrier wherein is a plasmid.

11. each carrier of claim 7 to 10, wherein the gene that can be operatively connected is a therapeutic nucleic acids.

12. each carrier of claim 7 to 11 comprises following any: SEQ IDNOs 1 or 2, CMV promotor, multiple clone site, polyadenylic acid sequence and the gene of coding selected marker thing under suitable controlling elements.

13. host cell comprises each polynucleotide of claim 1 to 6, or each carrier of claim 7 to 12.

14. each each carrier or the application that is used for gene therapy medicine in preparation of the host cell of claim 13 of polynucleotide, claim 7 to 12 of claim 1 to 6.

15. pharmaceutical composition, with the claim 1 to 6 of pharmaceutical excipient combination each polynucleotide, claim 7 to 12 each carrier or the host cell of claim 13.

16. claim 1 to 6 each polynucleotide, claim 7 to 12 each carrier or the host cell of claim 13 in cell culture system for obtaining the application of required gene product.

17.SEQ the application of the polynucleotide of ID NOs 1 or 2 in improving endogenous gene expression is included in the position that can be operatively connected endogenous gene polynucleotide is inserted in the genome of cell, thereby improves the expression of gene level.

18. claim 1 to 6 each polynucleotide, claim 7 to 12 each carrier or the host cell of claim 13 in the expression that obtains the inverted defined gene sequence with the application in the gene order of inactivation correspondence.

19. each each the application of polynucleotide in the preparation expression library of carrier of polynucleotide, claim 7 to 12 of claim 1 to 6.

20.SEQ the application of the polynucleotide of ID NOs 1 or 2 in the method for identifying the expressible gene in the non-human animal, comprise that the construct that will contain polynucleotide is inserted in non-human animal's the embryonic stem cell, wherein said construct can allow to carry out medicament selection after inserting the gene of expressing.

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