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WO2001009351A1 - Nouveaux vecteurs et systeme d'integration ciblee selectionnable de transgenes dans un chromosome sans marqueurs de resistance aux antibiotiques - Google Patents

Nouveaux vecteurs et systeme d'integration ciblee selectionnable de transgenes dans un chromosome sans marqueurs de resistance aux antibiotiques Download PDF

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WO2001009351A1
WO2001009351A1 PCT/US2000/021053 US0021053W WO0109351A1 WO 2001009351 A1 WO2001009351 A1 WO 2001009351A1 US 0021053 W US0021053 W US 0021053W WO 0109351 A1 WO0109351 A1 WO 0109351A1
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sequence
cell
sequences
vector
cassette
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Susan M. Rosenberg
Mary-Jane Lombardo
Laura Gumbiner-Russo
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Baylor College of Medicine
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Baylor College of Medicine
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination

Definitions

  • the invention relates to vectors and a method for creating a transgenic cell by negatively selecting cells deficient in integration of a transgene.
  • Gene expression and cloning in bacteria is almost exclusively done utilizing plasmids, and most of them are derived from multicopy ColE 1 plasmids such as pBR322 and its relatives. Expression of genes from plasmids causes multiple significant problems for in vivo experimental work. For instance, nearly all plasmids are maintained in cells using selection for antibiotic resistance, and for in vivo studies in which multiple mutations are also generated in host cells, one can exhaust the available resistance markers. Expression or overexpression of some genes can cause toxicity to the cell, or subsequent slow growth of the cell, and thus create a selection for the faster growing cells that lose the plasmid or for cells that accumulate plasmids containing a mutation that inactivates the gene.
  • plasmid genes such as helicases that can disrupt plasmid replication machinery components
  • subsequent slow growth of the cell thus creating a selection for the faster growing cells that lose the plasmid or for cells which accumulate plasmids containing a mutation which inactivates the gene.
  • Plasmids containing certain sequences such as chi sites that promote rolling circle replication of plasmids, direct or inverted repeats can render the plasmid unstable and subsequently lost from the cell.
  • antibiotics used for selection will interfere with purification and recovery of desirable protein products. Another problem with antibiotic selection is that the cost of antibiotics for selection of plasmids can be significant.
  • antibiotic resistance for plasmid selection is particularly troublesome with plasmids whose selectable marker is ampicillin resistance (most pBR322 derivatives); because the resistance is diffusable, most cells in a culture can survive without the plasmid because the medium is detoxified by a few plasmid-bearing cells.
  • plasmids replicated by rolling circle replication in particular desirable bacterial strains can result in loss of sequences for two reasons: (i) rolling circle replication can be allowed across inverted repeats present in hairpin structures; and (ii) long linear plasmid multimers made during rolling circle replication are unstable, which promotes plasmid loss even in the presence of an antibiotic.
  • plasmid may alternatively be maintained in a cell through complementation of an auxotrophic mutation on the chromosome, this approach severely restricts the composition of the fermentation medium to one lacking the required nutrient of the host bacteria. Moreover, syntrophism may allow cells to continue growth after loss of the plasmid.
  • Cloning vectors for integration of a plasmid into prophage sequences at the phage lambda origin of replication have been utilized (Boyd and Sherratt, 1995). Again, antibiotic resistance is the means for selection for integration.
  • One embodiment of the present invention is a method for creating a transgenic cell comprising the steps of: (1) introducing a linear vector into the cell wherein said vector contains a cassette comprising sequentially from 5' to 3': a 5' flanking sequence, a polylinker site containing a nucleic acid sequence of interest, and a 3' flanking sequence wherein said 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell where the nucleic acid sequence of interest is to be inserted; and (2) integrating said cassette into the chromosome through recombination between the flanking sequences and the homologous DNA sequences wherein said homologous DNA sequences flank a conditional killing module in a chromosome; and (3) negatively selecting against cells retaining said conditional killing module and deficient for integration of said linear vector.
  • the cassette further includes a 5' rare-cutter restriction site which is 5' to the 5' flanking sequence and a 3' rare-cutter restriction site which is 3' to the 3' flanking sequence wherein said 5' and 3' rare-cutter restriction sites have one or more sequences which are cut by rare cutter restriction enzymes.
  • the rare-cutter restriction sites are selected from the group consisting of Srfi, Ndel, Sfil, Avrll and Ascl.
  • the 5' and 3' flanking sequences comprise nucleic acid sequences which flank ⁇ ttB in Escherichia coli.
  • a method for creating a transgenic cell comprising the steps of: (1) introducing a linear vector into the cell wherein said vector contains a cassette comprising sequentially from 5' to 3': a 5' flanking sequence, a nucleic acid sequence of interest, and a 3' flanking sequence wherein said 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell where the nucleic acid sequence of interest is to be inserted; and (2) integrating said cassette into the chromosome through recombination between the flanking sequences and the homologous DNA sequences wherein said homologous DNA sequences flank a conditional killing module in a chromosome; and (3) negatively selecting against cells retaining said conditional killing module and deficient for integration of said linear vector.
  • the cassette is generated by polymerase chain reaction.
  • conditional killing module includes a conditional repressor.
  • conditional killing module contains temperate prophage sequences including a temperature sensitive conditional repressor.
  • cell is Escherichia coli and said conditional killing module is the lambda xisl clts857 prophage.
  • said cell is Escherichia coli and said conditional killing module is the lambda wi clts857 prophage, the defective lambda prophage lambda clts857 A(cro-bioA), the defective lambda prophage lambda clts857 clind A(cro-bioA), the defective lambda prophage lambdaxisl clts857 A(cro-bioA), the defective lambda prophage lambdaxisl clts857 clind A(cro-bioA), the defective lambda prophage lambda clts857 clind A(cro- ⁇ ttR), or the defective lambda prophage lambda clts ⁇ °57 clind P BAD A(cro- ⁇ ttR).
  • the integrated cassette is transduced by recombination between cells.
  • the integrated cassette is transduced between cells with the assistance of a helper phage.
  • said cell is Escherichia coli and said integrated cassette is transduced between Escherichia coli cells which contain the prophage lambda xisl clts857 with the assistance of a PI helper phage.
  • said cell is a proficient host for linear replacement using homologous recombination and is selected from the group consisting of recD, recBC sbcBC, recBC sbcA, recB carrying ⁇ red" genes and rec + carrying ⁇ red" and gam + genes.
  • Another specific embodiment is a vector selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.
  • An additional embodiment is the vector for introduction of a sequence of interest into a cell wherein said vector includes a cassette, said cassette comprising sequentially from 5' to 3': a 5' rare-cutter restriction site which is 5' to a 5' flanking sequence, a polylinker site containing the nucleic acid sequence of interest, and a 3' rare cutter restriction site which is 3' to the 3' flanking sequence wherein said 5' and 3' rare cutter restriction sites have one or more sequences which are cut by rare-cutter restriction enzymes and wherein said 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell where the nucleic acid sequence of interest is to be inserted.
  • the 5' and 3' flanking sequences of said cassette are sequences which flank attB in Escherichia coli.
  • the 5' and 3' rare cutter restriction sites are selected from the group consisting of Srfi, Ndel, Sfil, Avrll, and Ascl.
  • Another embodiment is the vector for introduction into a cell wherein said vector includes a cassette comprising sequentially from 5' to 3': a 5' rare cutter restriction site selected from the group consisting of Srfi, Ndel, Sfil, Avrll, and Ascl, a 5' flanking sequence, a polylinker site containing a nucleic acid sequence of interest, a 3' flanking sequence and a 3' rare cutter restriction site selected from the group consisting of Srfi, Ndel, Sfil, Avrll, and Ascl.
  • the 5' and 3' flanking sequences consist of nucleic acid sequences which flank attB in Escherichia coli.
  • nucleic acid sequence of interest is regulated by nucleic acid sequence selected from the group consisting of promoter sequence and bacterial translation- initiation sequence.
  • the promoter is an inducible promoter.
  • the promoter is the P BAD promoter.
  • the bacterial translation-initiation sequence is a Shine Dalgarno sequence.
  • Figure 1 depicts a strategy for linear replacement using the TGV system.
  • Figure 2 depicts the TGV-Light plasmid having SEQ ID NO: 1.
  • Figure 3 depicts the TGV-Cat plasmid having SEQ ID NO:2.
  • Figure 4 depicts the TGV-Kan plasmid having SEQ ID NO:3.
  • Figure 5 depicts the TGV-Tet plasmid having SEQ ID NO:4.
  • Figure 6 depicts the TGV-ProkExpress plasmid having SEQ ID NO:5.
  • Figure 7 depicts the TGV-Express plasmid having SEQ ID NO:6.
  • Figure 8 depicts the TGV-Express Cat plasmid having SEQ ID NO:7.
  • Figure 9 depicts the TGV-Express Kan plasmid having SEQ ID NO: 8.
  • Figure 10 depicts the TGV-Express Tet plasmid having SEQ ID NO:9.
  • Figures 11A and 11B demonstrate a comparison of efficiency of linear replacement TGV in different E. coli rec mutant genotypes.
  • Figure 11A shows a comparison of mono- versus multi-lysogens with and without the addition of pTGV-Light DNA.
  • Figure 1 IB shows a comparison of isogenic strains with and without the addition of pTGV-Light DNA.
  • Figures 12A and 12B demonstrate verification of linear replacement by colony PCR.
  • Figure 12A the presence of a PCR product was assayed.
  • Figure 12B the verification of PCR products was confirmed. DESCRIPTION OF THE INVENTION
  • blue-cutter restriction enzyme site is defined as a restriction enzyme site at which said enzyme cleaves one phosphodiester bond of each DNA strand between the same two specific hydrogen-bonded base pairs. Said cleavage produces DNA fragments with ends which contain no single stranded protrusion. Said sites may be of any size or form, including methylated form, and some examples include Smal, EcoKV, or Xmnl.
  • cell as used herein is defined as a structural unit of an organism, surrounded by a membrane and composed of cytoplasm and at least one nucleus or nucleoid. In some cells there may be a cell wall outside the membrane.
  • Cells as used herein can mean any kind of cells, including prokaryotic microorganisms, such as bacteria, and eukaryotic microorganisms, such as fungi, yeasts, algae, etc. Cells may also be of vegetable or animal (including human) origin.
  • condition killing module is defined as DNA sequence which contains (i) sequence encoding a gene product that will kill the cell under some conditions but not others.
  • sequence encoding a conditional repressor and a sequence whose expression is controlled by said conditional repressor and whose expression is lethal to the cell.
  • conditional repressor would work in the present invention.
  • conditional killing module contains prophage sequences which encode a gene product required for cell lysis and sequence which encodes a temperature sensitive repressor which represses expression of said cellular lysis gene product.
  • Other sequences which would be lethal in any form to the cell would be apparent to those skilled in the art.
  • DNA as used herein is defined as deoxyribonucleic acid.
  • 5' refers to a reference point for an entity such as a nucleotide or nucleotides which are in a position relative to a specific nucleotide or nucleotides through which there is linkage with the fifth carbon of 2-deoxyribose.
  • 5' flanking sequence as used herein is defined as a sequence which is 5' relative to a polylinker site which contains a nucleic acid sequence of interest.
  • 5' rare cutter restriction site is defined as a restriction enzyme site which is 5' relative to both a polylinker site which contains a nucleic acid sequence of interest and a 5' flanking sequence.
  • homologous recombination as used herein is defined as the replacement of one sequence with a homologous sequence.
  • linear replacement as used herein is defined as homologous recombination between a linear DNA fragment and a chromosomal site.
  • lysogen as used herein is defined as a bacteria which contains viral genetic material within its host genome.
  • the term "negatively selecting" as used herein is defined as the act of using a specific condition to kill, or prevent from growing, cells with a particular phenotype while permitting survival and growth of cells without said particular phenotype.
  • the phenotype of the preferred embodiment of the invention is the presence of prophage sequences containing a temperature sensitive conditional repressor which represses gene products required for cell lysis.
  • non blunt-cutter restriction enzyme site is defined as a restriction enzyme site at which said enzyme cleaves one phosphodiester bond of each DNA strand between two different specific hydrogen-bonded base pairs. Said cleavage produces DNA fragments with ends which contain either a 5' or 3' single stranded protrusion. Said sites may be of any size or form, including methylated form, and some examples include EcoRI, Sac I, or Sal I.
  • P BAD as use d herein is defined as the arabinose inducible promoter.
  • PCR as used herein is defined as polymerase chain reaction. Methods regarding all aspects of PCR, including reaction conditions and primer design, are well known in the art.
  • polylinker site as used herein is defined as a site containing at least one restriction enzyme site for the purpose of inserting a nucleotide sequence of interest.
  • restriction enzyme sites would be useful for such a purpose.
  • Said restriction enzyme site can include any DNA sequence which is recognized by a specific restriction enzyme and could be a blunt- cutter restriction enzyme site or a non blunt-cutter restriction enzyme site.
  • prophage as used herein is defined as a virus which has incorporated its genetic material into the cell or genome of a host.
  • rare cutter restriction enzyme as used herein is defined as a restriction enzyme which cuts DNA at a rare cutter restriction site. Some examples include Srfi, Ndel, Sfil, Avrll, and Ascl.
  • IR restriction site is defined as a restriction site which is present in the DNA of the cell used at a frequency no greater than one in every
  • restriction sites themselves can be of any size or form, including a methylated form or a blunt cut site.
  • repressor as used herein is defined as an agent which represses expression of a particular DNA sequence.
  • RNA as used herein is defined as ribonucleic acid.
  • sbc supressor of RecBCD.
  • sertrophism as used herein is defined as the act of two entities (proteins, nucleic acids, cells, etc.) which can not perform a function independently, but acting together can perform the function.
  • emperate prophage as used herein is defined as a virus which infects a bacteria cell and whose genetic material can become integrated into the host genome, therein duplicated along with the host material upon replication.
  • the viral DNA has the capacity to educt from the host genome which subsequently leads to cellular lysis.
  • 3' refers to a reference point for an entity such as a nucleotide or nucleotides which are in a position relative to a specific nucleotide or nucleotides through which there is linkage with the third carbon of 2-deoxyribose.
  • 3' flanking sequence as used herein is defined as a sequence which is 3' relative to a polylinker site which contains a nucleic acid sequence of interest.
  • 3' rare cutter restriction site as used herein is defined as a restriction enzyme site which is 3' relative to both a polylinker site which contains a nucleic acid sequence of interest and a 3' flanking sequence.
  • transgene as used herein is defined as a foreign gene introduced into the genome of the host or a native gene of the host introduced into a new position in the host.
  • transgene is used herein interchangeably with the term “nucleic acid sequence of interest.”
  • vector as used herein is defined as any vehicle which delivers a nucleic acid into a cell. In a preferred embodiment, said vector is a linear DNA fragment.
  • One embodiment of the present invention is a method for creating a transgenic cell comprising the steps of: (1) introducing a linear vector into the cell wherein said vector contains a cassette consisting of sequentially from 5' to 3': a 5' flanking sequence, a polylinker site containing a nucleic acid sequence of interest, and a 3' flanking sequence wherein said 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell at a location where the nucleic acid sequence of interest is to be inserted; and
  • the cassette can further include a 5' rare cutter restriction site which is 5' to the 5' flanking sequence and a 3' rare cutter restriction site which is 3' to the 3' flanking sequence wherein said 5' and 3' rare cutter restriction sites have one or more sequences which are cut by rare cutter restriction enzymes.
  • said conditional killing module includes a conditional repressor.
  • conditional killing module contains temperate prophage sequences including a temperature sensitive conditional repressor.
  • cell is Escherichia coli and said conditional killing module is the lambda xisl clts857 prophage.
  • said cell is Escherichia coli and said conditional killing module is the lambdaxisl clts857 prophage, the defective lambda prophage lambda clts857 A(cro-bioA), the defective lambda prophage lambda clts857 clind A(cro-bioA), the defective lambda prophage lambda xisl clts857 A(cro-bioA), the defective lambda prophage lambdaxisl clts857 clind ' A(cro-bioA), the defective lambda prophage lambda clts857 clind A(cro- ⁇ ttR), or the defective lambda prophage lambda clts857 clind P BAD A(cro- ⁇ ttR).
  • the integrated cassette is transduced between cells.
  • the integrated cassette is transduced between cells with the assistance of a helper phage.
  • said cell is Escherichia coli and said conditional killing module is transduced between Escherichia coli cells which contain the prophage lambda xisl clts857 with the assistance of a PI helper phage.
  • said cell is a proficient host for linear replacement using homologous recombination and has a genotype selected from the group consisting of recD, recBC sbcBC, recBC sbcA, recB carrying ⁇ red" genes and rec + carrying ⁇ red" and gam + genes.
  • Another specific embodiment is a vector selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.
  • An additional embodiment is the vector for introduction of a sequence of interest into a cell wherein said vector includes a cassette, said cassette comprising sequentially from 5' to 3': a 5' rare-cutter restriction site which is 5' to a 5' flanking sequence, a polylinker site containing the nucleic acid sequence of interest, and a 3' rare-cutter restriction site which is 3' to the 3' flanking sequence wherein said 5' and 3' rare-cutter restriction sites have one or more sequences which are cut by rare restriction enzymes and wherein said 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell where the nucleic acid sequence of interest is to be inserted.
  • the 5' and 3' flanking sequences of said cassette are sequences which flank attB in E. coli.
  • the 5' and 3' rare-cutter restriction sites are selected from the group consisting of Srfi, Ndel, Sfil, Avrll, and Ascl.
  • Another embodiment is the vector for introduction into a cell wherein said vector includes a cassette comprising sequentially from 5' to 3': a 5' rare-cutter restriction site selected from the group consisting of Srfi, Ndel, Sfil, Avrll, an Ascl, a 5' attB flanking sequence, a polylinker site containing a nucleic acid sequence of interest, a 3' attB flanking sequence and a 3' rare-cutter restriction site selected from the group consisting of Srfi, Ndel, Sfil, Avrll, and Ascl.
  • a 5' rare-cutter restriction site selected from the group consisting of Srfi, Ndel, Sfil, Avrll, an Ascl
  • a 5' attB flanking sequence a polylinker site containing a nucleic acid sequence of interest
  • 3' attB flanking sequence a 3' rare-cutter restriction site selected from the group consisting of Srfi, Ndel, S
  • flanking sequences and 3' flanking sequences incorporated in the vector for homologous recombination would have to be homologous to sequences present within the host genome and each flanking sequence must be at least about 23 base pairs of DNA.
  • identity of the sequence could be any sequence so long as loss of the interstitial sequence beween said 5' and 3' flanking sequences upon its removal through integration of the transgene would not be lethal to the cell.
  • increasing the size of sequence utilized in the vector for homologous recombination would increase the likelihood of recombination.
  • flanking sequence to use for homologous recombination.
  • sequences which flank either side of attB in the E. coli genome are utilized in the present invention as the 5' flanking sequences and 3' flanking sequences. These sequences are provided herein as SEQ ID NO:34 and SEQ ID NO:35.
  • SEQ ID NO:34 and SEQ ID NO:35 are provided herein as SEQ ID NO:34 and SEQ ID NO:35.
  • any region within these sequences may be utilized for homologous recombination so long as they meet the conditions mentioned above.
  • the linear vector is a PCR-generated nucleic acid
  • any sequence within these sequences may be utilized to design primers for polymerization, and a skilled artisan would be aware how to design the primers and what parameters should be considered.
  • the vector backbone which contains the cassette of the invention may be any nucleic acid sequence which would allow liberation of said cassette upon restriction enzyme digestion with a rare-cutter restriction site of said cassette or upon PCR or other means of liberation of the cassette.
  • a linear fragment for introduction into a cell is generated by polymerase chain reaction by methods well known in the art.
  • the primers utilized for the polymerase chain reaction include sequence which derives from either 5' or 3' flanking sequences, such as those which flank attB in the Escherichia coli genome, and sequence which derives from the nucleic acid sequence of interest.
  • the nature of the transgene to be integrated can be any DNA fragment which would be useful to make a transgenic organism.
  • a skilled artisan would be fully aware how to generate the vector constructs using standard molecular biology methods well known in the art and could use discretion regarding what sequences would be useful to insert into the genome of the host organism.
  • a skilled artisan would also know that in addition to inserting DNA sequences which produce a useful protein as a final gene product that DNA sequences which produce a useful RNA as a final gene product may be inserted.
  • control sequences required for expression of said transgene including promoters, enhancers, or any cis- acting elements required for regulation of expression may be employed.
  • any sequences necessary for production of the final gene product for instance Shine- Dalgarno sequences or AUG initiator codons, may be included in the cassette.
  • Shine-Dalgarno sequence is, in a specific embodiment, part or all of the polypurine sequence AGGAGG and is present just prior to an AUG initiation codon.
  • the sequence is associated with binding of a ribosome to mRNA and in a specific embodiment is complementary to a sequence at the 3' end of 16S rRNA. Based on the gene of interest, one skilled in the art would know which vectors and regulatory elements would be useful.
  • the present invention provides a conditional repressor to repress genes whose expression would be lethal to the cell.
  • the nature of the activity of the conditional repressor is temperature sensitive.
  • a cl gene product serves as the conditional repressor.
  • any other ⁇ cl gene that produces a functional repressor can be used.
  • Other repressor genes such as, for example, the ⁇ cro gene can also be used. It would be known to those skilled in the art that repressors responsive to other conditions, for instance changes in light, chemicals, osmolarity, pressure, touch, sound, stress, or concentration or nature of an associated protein may be used.
  • said repressor is a nonsense repressor.
  • a nonsense repressor is a gene product which allows the insertion of an amino acid into an extending polypeptide chain in response to a nonsense codon.
  • a nonsense codon is a chain-terminating codon introduced as a result of a nonsense mutation such as a base substitution or frameshift mutation.
  • the sequence of said conditional killing module is a sequence containing a lethal nonsense mutation and wherein said nonsense repressor is a conditional nonsense repressor.
  • a preferred embodiment of the present invention utilizes the arabinose- inducible P BAD promoter in the vectors described in Examples 9, 10 and 11, a skilled artisan would be aware of other promoter or promoter derivatives or fragments thereof which would have the same function of providing an element for inducibility of expression of a nucleotide sequence.
  • Other examples of inducible promoters are the galactose promoter, the lac, tac, or mac promoters and the Pspac promoter.
  • a variety of methods for introducing vectors into host cells are known in the art, including but not limited to electroporation; transformation employing calcium chloride, rubidium chloride calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is an infectious agent, such as a retro viral genome).
  • a linear fragment generated by polymerase chain reaction is introduced into a cell, wherein the fragment comprises a 5' flanking sequence, a nucleic acid sequence of interest, and a 3' flanking sequence, wherein the 5' and 3' flanking sequences are homologous to a sequence in a chromosome in the cell at a location where the nucleic acid sequence of interest is to be inserted; integration of the cassette into the chromosome through recombination occurs between the flanking sequences and the homologous sequences, wherein the homologous DNA sequences flank a conditional killing module in the chromosome; and negatively selecting against cells retaining the conditional killing module and deficient for integration of the cassette.
  • the linear DNA for introduction is a polymerase chain reaction product generated with one primer containing approximately
  • the primer contains flanking sequence anywhere in the range from about 15 to about 30 nt and the primer contains sequence from a gene of interest in the range from about 10 to about 20, although other lengths would also work.
  • other parameters are important for design including the content of G's and C's, the melting temperature of the primer and the presence of any sequence which may facilitate undesirable secondary structure within the primer or with another primer.
  • the vector comprises a 5' flanking sequence, a nucleic acid sequence of interest, an antibiotic resistance gene, and a 3' flanking sequence.
  • the prophage/conditional killing module system of the present invention is not required. Instead, the cassette integrates into the sequences which correspond to the 5' and 3' flanking sequence, and the antibiotic resistance is selected for to identify transgenic cells which have incorporated the nucleic acid sequence of interest.
  • Bacteriophage ⁇ is a temperate bacteriophage which upon infection of Escherichia coli follows either of two mutually exclusive cycles.
  • the bacteriophage DNA replicates autonomously, directs synthesis and assembly of bacteriophage components, and kills the cells concomitant with the release of mature bacteriophage.
  • the bacteriophage becomes integrated into the host chromosome as a prophage, replicates on the chromosome with the endogenous host DNA, and blocks synthesis of bacteriophage components.
  • a bacteriophage gene, ⁇ cl codes for a repressor that maintains the lysogenic state and blocks expression of genes for bacteriophage components and maturation. If the repressor is inactivated or is no longer present in the cell the prophage educts from the chromosome, enters the lytic cycle, and subsequently kills the cell. Bacteriophage with a defective ⁇ cl gene can not maintain the lysogenic state and are lethal to the cell.
  • a temperature sensitive allele of the ⁇ cl gene, ⁇ clts857 allows repression at lower permissive temperatures but loses repressor activity at higher non-permissive temperatures.
  • a temperature shift to higher temperatures lyses the cells by inducing the lytic cycle of the lambda prophage which, in accordance with the present invention, has been incorporated into the host cell strain.
  • a temperature sensitive repressor which represses a lethal sequence that causes host cell death is used and when the host cells are cultured at a temperature which inactiviates the repressor and, at a temperature which is not within the range for permissive culture, the present invention provides cellular death.
  • the prophage is the temperate bacteriophage ⁇ residing within the host chromosome of Escherichia coli, the temperature sensitive repressor repressing expression of ⁇ prophage sequences is ⁇ clts857, and the temperature shift to greater than 34° C inactivates said repressor.
  • conditional killing module includes a conditional repressor.
  • conditional killing module contains temperate prophage sequences including a temperature sensitive conditional repressor.
  • cell is Escherichia coli and said conditional killing module is the lambda xisl clts857 prophage.
  • said cell is Escherichia coli and said conditional killing module is the lambdaxzsi clts857 prophage, the defective lambda prophage lambda clts ⁇ °57 A(cro-bioA), the defective lambda prophage lambda clts ⁇ °57 clind ' A(cro-bioA), the defective lambda prophage lambdaj ⁇ J clts857 A(cro-bioA), the defective lambda prophage lambdaxisl clts857 clind A(cro-bioA), the defective lambda prophage lambda clts857 clind ' A(cro- ⁇ ttR), or the defective lambda prophage lambda clts ⁇ °57 clind P BAD A(cro- ⁇ ttR).
  • the present invention provides significant improvements over the present art by describing vectors in which transgenes and flanking DNA are inserted in the chromosome by linear replacement via homologous recombination in a cell carrying a prophage with a conditional allele of a repressor.
  • said condition is an increase in temperature.
  • the present invention uses as a mode of negative selection the system of repression of lethal gene products by said conditional repressor, in marked contrast to the presence of a marker, such as an antibiotic resistance marker, which is one of the significant improvements the present invention has over the related art.
  • a marker such as an antibiotic resistance marker
  • the TGV system employs a simple two-step method of cloning and homologous replacement.
  • Each vector contains a multiple-cloning site (MCS) flanked by approximately 2 kb of sequence from the attB region of the E. coli chromosome (Fig. 1).
  • MCS multiple-cloning site
  • the multiple cloning site replaces 23 bp of the attB core sequence (Weisberg and Landy, 1983) with eight restriction endonuclease sites for cloning transgenes (Fig . 1 ) .
  • the gene being transferred is cloned into this multiple cloning site.
  • the vector now containing the transgene (nucleic acid sequence of interest), is linearized using one of the five infrequently cutting restriction enzymes that cut in the rare-cutter sites (RCS) at the outside ends of the entire region of homology (Fig. 1).
  • This linear fragment is then transformed into recombination-proficient E. coli carrying a lambda prophage, which is excision-defective (xisl) and carries a temperature-sensitive Cl repressor (clts857) such that the prophage is quiescent at 34°C or lower, but is induced and kills the cell at high temperature (42°C) .
  • Transformants are selected by plating at the restrictive temperature (42°C).
  • MCS multiple cloning site (such as Sph I, Xbal, Xhol, BgHl, Hindl ⁇ l, CM, Sna l, and Agel) and RCS stands for rare cutter sites (such as Ascl, Avrll, Sfil, Ndel, and Srfi).
  • pTGV-Light is the simplest version containing the regions of homology, Table 1.
  • pTGV-ProkExpress is pTGV-Light with an arabinose-inducible promoter (P BAD ) (Guzman et al, 1995) added just upstream of the multiple cloning site, to allow regulatable expression of the transgene.
  • the AraC regulatory protein is expressed from its normal chromosomal location, although in a specific embodiment it may be provided on a TGV vector or another vector.
  • pTGV-Express like pTGV-ProkExpress, contains P BAD and also includes the Shine-Dalgarno prokaryotic translation-initiation sequence added between the P BAD promoter and the multiple cloning site for expression of heterologous genes in E. coli.
  • Versions of pTGV-Light and pTGV-Express were also constructed with genes encoding chloramphenicol-resistance, kanamycin-resistance, or tetracycline-resistance so that selection for antibiotic resistance may be used if desired. No prophage is necessary when antibiotic selection is used.
  • Sites not available in these vectors are as follows: Agel in pTGV-ProkExpress, pTGV-Express, pTGV-Express CAT, and pTGV-Express Tet; Htndlll, CM, and Xhol in pTGV-Kan; Sphl in pTGV-Tet; and Agel, CM and Xhol in pTGV-Express Kan. All sites are separated by one or two nucleotides. Plasmid sequences are available.
  • EXAMPLE 2 The TGV System: TransGenic Escherichia coli Sectors for chromosomal gene expression
  • the rare-cutter restriction site is selected such that digestion with the rare-cutting enzymes will not cut whatever transgene(s) of interest will be inserted into a polycloning site that was engineered into the center of the bacterial homology, replacing the attB site itself.
  • the transgenes and flanking DNA are placed in the Escherichia coli chromosome by homologous recombination as follows: the bacterial DNA region, of 3 kilobases plus inserted transgenes, is amplified by PCR with primers complementary to the ends of the bacterial segment to produce a linear fragment or alternatively is liberated by restriction enzyme digestion of a plasmid preparation. This linear fragment is elecfroporated into Escherichia coli cells which are recD, recBC sbcBC, recBC sbcA, recB strains carrying ⁇ red" genes, or rec + strains carrying ⁇ red" and gam + genes.
  • recD Cells which are recD do not degrade linear DNA, and linear replacement-recombination reactions work efficiently in this background (Russell et ⁇ /.,1989).
  • Linear replacement in recD cells has been used previously to construct several useful alleles (e.g. seeRazavy etal, 1996).
  • the recD cell is a ⁇ lysogen whose prophage carries a temperature-sensitive repressor allele, clts857. Such a prophage is lysogenic at 32° but becomes lytic at temperatures greater than 34° and kills the cell (Murray, 1983).
  • Selection for linear replacements of the prophage occurs by plating the elecfroporated cells at 42° following a suitable low temperature incubation period to let the replaced chromosome segregate from prophage-bearing sister chromosomes.
  • the cells may also carry a ⁇ -resistance mutation so that they are not killed by any phage in the culture on loss of their prophage, and the prophage is also excision defective due to the xisl allele.
  • Methods concerning similar lysogens and their selection are known in the art (e.g. Rosenberg et al, 1985; Rosenberg, 1985; Rosenberg et al, 1985 ; Rosenberg, 1987). Colonies that survive high temperature have incorporated the nucleic acid sequence of interest, which had been inserted into the polycloning site of the vector.
  • TGV plasmids are derivatives of pLGR2, a pBR322-derived-ampicillin resistant plasmid that contains approximately 2 kilobases of DNA flanking both sides of the bacteriophage lambda attachment site, attB, (SEQ ID NO : 34 and SEQ ID NO : 35) and has sites for infrequently-cutting restriction enzymes flanking the entire 4kb of insert DNA.
  • pLGR2 was constructed from pWRl 01 (a pBR322-based plasmid carrying the Escherichia coli attB site within a 1.7 kb EcoRI-Zt ⁇ /wHI insert) as below.
  • TGV plasmids that were constructed using PCR-generated DNA are sequenced. Methods well known in the art were utilized for cloning (Sambrook et al, 1989 The template for PCR was either the appropriate purified plasmid DNA (such as for resistance genes) or the bacterial chromosome (such as in colony suspensions). Bacterial strain MG1655 (Bachman, 1996) was used as chromosomal template in all cases, except in construction of pMJl in which W3110 (Bachman, 1996) was used, and in construction of the original TGV-Light polylinker.
  • Additional chromosomal flanking sequence was placed on the BamHl side of the pWRlOl insert by ligating an Ndel-BamHl fragment generated by PCR into Ndel- if ⁇ mHI-digested p WRl 01 to create pMJ 1.
  • the PCR primers amplify the Escherichia coli bio region.
  • One primer (5'- TCCGGTCTTCATATGCAGCAACGTGCT-3'; SEQ ID NO: 10) creates an Ndel site for cloning and the other (5'- AAGGCCGAATCCAGACA- 3'; SEQ ED NO:l 1) contains a natural Ban ⁇ l site.
  • Sites for the rare-cutter restriction enzymes Srfi, Ndel, Sfil, Avrll, and Ascl were added by ligating annealed oligonucleotides (5'- TATGGCGCCTAGGCCAATTGGGCCCGGGCA-3' (SEQ ID NO: 12) and 5'-'TATGCCCGGGCCCAATTGGCCTAGGCGCGCCA-3' (SEQ ID NO: 13) containing those sites and Ndel-compatible overhangs into the Ndel site of pMJl , creating pLGRl .
  • oligonucleotides 5'- TATGGCGCGCCTAGGCCAATTGGGCCCGGGCA-3' (SEQ ID NO: 12) and 5'-'TATGCCCGGGCCCAATTGGCCTAGGCGCGCCA-3' (SEQ ID NO: 13) containing those sites and Ndel-compatible overhangs into the Ndel site of pMJl , creating pLGRl .
  • EXAMPLE 4 Construction of pLGR2 Additional chromosomal flanking sequence and sites for the "rare-cutter" enzymes Srfi, Ndel, Sfil, Avrll, and Ascl were added to the Ec ⁇ RI side of the insert by ligating an EcoRI-Ec ⁇ O190I fragment generated by PCR using primers 5'- GTGAGTATCAGGGAACGGTA-3' (S ⁇ Q ID NO: 14; t h e ⁇ c o R I s i t e i s a n t u r a l s i t ) a n d 5 ' - GAGCTGACAGAGGCCCTGGCGCGCCTAGGCCATATGGGCCCGGGCGAGC ATATTGATCCGCTGCAAACTGAA-3' (S ⁇ Q ID NO:15; creating an EcoO190I site and the above rare-cutter sites) into EcoRI-EcoO190I digested pMJl .
  • TGV-Light (Figure 2) was constructed by replacing the attB core sequence of pLGR2 with a polylinker.
  • the polylinker was created by PCR with four primers : primer A 5'- GAGGTACCAGGCGCGGTTTG-3' (SEQ ID NO: 16); primer B 5'- GCACCGGTACGTATCGATAAGCTTAGATCTCTCGAGT-3' (SEQ ID NO: 17); primer C 5'- ACGTACCGGTGCGAAACGGGAAGGT-3' (SEQ ID NO: 18); and primer D 5'- GACGCGTACCGACTTTGG-3' (SEQ ID NO: 19) used in a two step PCR procedure with pMJl template DNA to create a final product with Kpnl and Mlul sites on the ends and a polylinker in place of the attB core sequence.
  • Primers A (SEQ ID NO: 16) and B (SEQ ID NO: 17) were used in one PCR reaction and primers C (SEQ ID NO : 18) and D (SEQ ID NO : 19) in a separate reaction.
  • the two resulting PCR products were used as template in a final reaction with primers A (SEQ ID NO: 16) and D (SEQ ID NO: 19). Sequence overlap between the two products allows generation of the final product which was digested with Kpnl and Mlul and ligated into Kp l-Mlul digested pLGR2.
  • the resulting plasmid is identical to pLGR2 except that the attB core sequence is replaced with the polylinker sequence.
  • This original polylinker was later replaced with anew one with additional nucleotides between some sites to enhance cleavage efficiency.
  • the replacement polylinker was ligated into the outermost polylinker sites Sphl and Agel as annealed oligonucleotides 5'-GC ATGCGTCTAGAGCTCGAGGTAGATCTGAAAG CTTGAATCGATGTACGTACTATACCGGT-3' (SEQ ID NO:20) and 5'-ACCGGTAT AGTACGTACATCGATTCAAGCTTTCAGATCTACCTCGAGCTCTAGACGCAT GC-3' (SEQ ID NO:21). See Figure 2 for the polylinker sites.
  • TGV-Cat ( Figure 3) was constructed by ligating the chloramphenicol-resistance cassette (chloramphenicol acetyl transferase, or cat gene) from pCAT19 (Fuqua, 1992) as a Sphl-Xbal fragment into Sphl-Xbal digested TGV-Light.
  • the polylinker in an early version of this construct was also replaced with the same annealed oligonucleotides (SEQ ID NO:20 and SEQ ID NO:21) as for TGV-Light, but digested with ⁇ l and Agel into the Xb ⁇ l-Agel sites to give TGV-Cat.
  • TGV-Kan ( Figure 4) was constructed by ligating the kanamycin-resistance gene from pUC4K (Vieira and Messing, 1982) as a Nspl digested PCR product into Sphl digested pTGV-Light using primers 5' TCAACATGTGTCTGCCTCGTGAAGAAG 3' (SEQ ID NO:22) and 5' TCAGCATGCAGCCAGGTTGTGTCTCAA 3' (SEQ ID NO:23) to create the Nspl and Sphl sites.
  • pTGV-Tet ( Figure 5) was constructed by ligating the tetracycline resistance (tet) gene from pACYC184 (Chang and Cohen, 1978) as a Styl-Xbal digested PCR product i n t o Xb a l d i g e s t e d p T G V - L i g h t u s i n g p r i m e r s 5 ' TCATCTAGATTAATGCGGTAGTTTATC 3' (SEQ ID NO:24) and 5' TCACCTAGGTGCAGCAGCAGTCGCTTC 3' (SEQ ID NO:25) to create the Styl and Xbal sites. Orientation was determined by digestion with Sphl. The presence of a Sphl site in the tet gene excludes its use for cloning.
  • TGV-Light vectors are best used when expressing a gene from its natural promoter. However, for overexpression work regulatable promoters would be advantageous (see also Example 10 and 11). Therefore, the P BAD inducible promoter has been included in the described vectors.
  • TGV-ProkExpress ( Figure 6) contains the P BAD promoter and the araC gene (encoding the AraC regulatory protein) amplified from the bacterial chromosome using primers 5'- CAGTCAGCTAGCTCCCGCCATTC-3' (SEQ ID NO:26) and
  • polylinker in this plasmid was also replaced using the same annealed oligonucleotide polylinker as pTGV-Light but cut with & ⁇ l and SnaSl.
  • the presence of an Agel site in the promoter excludes its use for cloning.
  • the vectors described in Examples 10 and 11 also provide a prokaryotic translation-start signal.
  • TGV-Express ( Figure 7) contains the P BAD promoter and the araC gene, but also contains an initiating methionine and a Shine-Dalgarno sequence (prokaryotic translation initiation sequence) upstream of the Met codon to allow efficient translation.
  • the P BAD promoter and araC were amplified from the bacterial chromosome by PCR with primers 5'- TCACATGTCTGAGCTCTCCCGCCATTCAGAGAAGAAAC-3' (SEQ IDNO:28; creating an Nspl site and a Sad site) and
  • pTGV-Express-CAT (Fig. 8), -Express-Kan (Fig. 9), and -Express-Tet (Fig. 10) were constructed using the cat, kan, or tet genes as Sphl fragments from the appropriate pKRP plasmid (Reece and Phillips, 1995).
  • Annealed oligonucleotides which contained a Sphl compatible end and a Sad end to give products with Sad overhangs (5' ACAGGAGCT 3' (SEQ ID NO:30) and 5' CCTGTCATG 3' (SEQ ID NO:31)), were added to each Sphl digest.
  • Escherichia coli cells were elecfroporated with either no DNA or with 1.5 ⁇ g
  • the basic vector, pTGV-Light was used without an insertion to test for replacement of the prophage using the anti- ⁇ prophage selection.
  • Linearized pTGV-Light was elecfroporated into a recombination-proficient strain carrying the temperature-sensitive prophage, and transformants were selected at 42°C. Although some colonies appeared in control platings of cells that were mock-transformed (without DNA), ten- to thirty-fold more appeared in platings of cells with DNA (Fig. 11a and l ib). The colonies on the control plates may result from reversion of the cits and subsequent survival of lysogens at high temperature. Replacement of the prophage was confirmed by polymerase chain reaction (PCR) across the multiple cloning site (Fig. 12a and 12b) as follows.
  • PCR polymerase chain reaction
  • PCR was performed using primers flanking attB to determine whether the prophage was absent (short PCR product) or not ( ⁇ prophage of 48 kilobases is too large to PCR across).
  • Ten transformants from the transformation with TGV-Light DNA and ten colonies from a control transformation with no DNA were plated at 42°C and subsequently were transferred into 50 ⁇ l water. These cell suspensions were used to provide template DNA for PCR with attB primers attBL: 5'-GGATTCGGTGTTATCG-3' (SEQ ID NO: 32) and attBR: 5'-GGATCCGGCCTTTTG-3'(SEQ ID NO:33).
  • FIG. 12 A PCR products were run on a 1 % agarose gel. Lanes 1-10 represent colonies picked from mock transformed plates. Lanes 11-20 represent colonies picked from plates transformed with linear pTGV-Light. The PCR product is approximately
  • the PCR products generated above were digested with restriction enzymes that cut the polylinker which should be present in the replaced DNA, but would not cut wild-type bacterial attB region DNA. This distinguishes the unlikely possibility that the temperature-resistant colonies obtained after transformation with TGV-Light DNA was simply cured of their prophage, which would return them to the natural attB region sequence.
  • Ten ⁇ l of each transformant PCR reaction was digested with BgM, a restriction enzyme whose site is present only in the polylinker of the 4.2 kilobase pair TGV linear replacement fragment. Digestion of the 1.75 kilobase pair PCR product results in 966 base pair and 783 base pair fragments.
  • PCR products from lanes 11-20 in IB were digested with Bgl ⁇ l (+) and run with uncut samples (-) on a 1.5% agarose gel. Digest products migrate as expected for 955bp and 775bp. Digestion of this PCR product with a restriction enzyme that cuts in the multiple cloning site yielded products of the correct size (Fig. 12b).
  • isogenic strains were compared in the mono-lysogenic form (Fig.1 IB). Each bar represents an average of four experiments performed on different days with different batches of competent cells. rec + was done separately from the others in three experiments, but in parallel with recBC sbcBC. A subset of these transformants was confirmed by PCR. Strains used (from left to right) are SMR5078, SMR5080, SMR5076, SMR5220, SMR5221, SMR5078, and SMR5449. Error bars represent one standard error of the mean. All strains showed a similar transformation efficiency, except for recD and rec + which were lower.
  • the recBC sbcBC strain was used as a mono-lysogen and a non-lysogen to compare the efficiencies of anti- ⁇ prophage versus drug selection.
  • the same number of molecules of linearized pTGV-CAT was elecfroporated into each strain, and selection was performed either against the prophage (at 42°C) or for the presence of the chloramphenicol resistance gene (Table 3).
  • Recovery of antibiotic resistant transformants was four- fold higher than recovery of transformants after selection against the prophage.
  • the strains in Table 4 also carry the AaraBAD Aim (Haldimann et al, 1998) deletion.
  • the AraC regulatory protein is expressed from its normal chromosomal location. Electro-competent cells were prepared (Sambrook et al, 1989) and were diluted 1:200 from LBH cultures into LBH plus 0.2% arabinose or 0.2% glucose followed by 5-6 hours of growth before harvesting. Following electro-transformation of a mix of 40-80ng of each plasmid, transformants were counted by dilution and plating Table 4. Inducible expression of a transgene inserted with TGV-Express.
  • Lambda lysogen deri tested using standard lambda methods (Murray, 1983). Mono- versus multi-lysogeny was determined using lambda cl90cl 7 as described (Shimada et al, 1972). Lambda xisl clts857 is ⁇ SR446.
  • SMR5075 are derived from C600 (Bachmann, 1996) and also contain thi, thr, leu, and supE. SMR423 also contains trp::Tn5 and supF.
  • TGV-Light and TGV-CAT were linearized by digestion with Ndel (or an appropriate enzyme) overnight at 37°C.
  • Shrimp alkaline phosphatase was included in the reaction mixture and was inactivated following digestion by incubation at 65°C for 20 minutes. 1 ⁇ g of the linear fragment was delivered to electrocompetent cells by electroporation (according to manufacturer's instructions for Bio-Rad E. coli Pulser) and allowed to recover for one hour in SOC medium (Sambrook et al, 1989) at 32°C.
  • Transformed cells were then concentrated, and either one-half or a dilution of the entire transformation mixture was plated on LBH27 with 20mM sodium citrate to inhibit infection of recombinants by free lambda phage. The plates were incubated at 42°C overnight. TGV-CAT transformations were treated similarly but plated on LBH with 25 ⁇ g/ml chloramphemcol, and were incubated overnight at 37°C.
  • Verification of linear replacement was done by colony PCR on transformants using primers attBL (5' GGATTCGGTGTTATCG 3'; SEQ ID NO:32) and attBR (5' GGATCCGGCCTTTTG 3'; SEQ ID NO:33) which anneal at sequences -900 and -700 bp from the core att site respectively.
  • primers attBL 5' GGATTCGGTGTTATCG 3'; SEQ ID NO:32
  • attBR 5' GGATCCGGCCTTTTG 3'; SEQ ID NO:33
  • Cloning and expression of genes in single copy in the bacterial chromosome is sometimes required to avoid the complications encountered with plasmids.
  • gene overexpression from plasmids can produce aberrant phenotypes not representative of the normal function of the gene product.
  • a PI lysate made from the strain containing the transgene is then used to transduce the gene into the desired lysogenized background, selecting again at the non-permissive temperature. This method allows expression of transgenes in a multitude of genetically relevant backgrounds.

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Abstract

L'invention concerne un procédé de production d'une cellule transgénique consistant: à introduire un vecteur linéaire dans la cellule, ledit vecteur contenant une cassette comportant séquentiellement de 5' à 3': une séquence flanquée 5', un site polylieur contenant une séquence d'acide nucléique d'intérêt, et une séquence flanquée 3', ces séquences flanquées 5' et 3' étant homologues à une séquence dans un chromosome de la cellule dans laquelle la séquence d'acide nucléique va être insérée, puis à intégrer cette cassette dans le chromosome par une recombinaison des séquences flanquées et des séquences ADN homologues dans laquelle lesdites séquences ADN présentent sur leurs flancs un module tueur conditionnel dans un chromosome, et enfin à sélectionner négativement contre les cellules qui retiennent ce module tueur conditionnel et qui sont déficientes sur le plan de l'intégration du vecteur linéaire.
PCT/US2000/021053 1999-08-02 2000-08-02 Nouveaux vecteurs et systeme d'integration ciblee selectionnable de transgenes dans un chromosome sans marqueurs de resistance aux antibiotiques Ceased WO2001009351A1 (fr)

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WO2004069996A3 (fr) * 2003-02-05 2004-12-09 Degussa Bacteries et procede de production de composes chimiques au moyen desdites bacteries i
WO2006025702A1 (fr) * 2004-09-02 2006-03-09 Korea Advanced Institute Of Science And Technology Fragment d'adn lineaire pour deletion sans marqueur, nouvelle souche presentant une formation de biofilm inhibee et methode de preparation correspondante
WO2008142028A1 (fr) * 2007-05-17 2008-11-27 Boehringer Ingelheim Rcv Gmbh & Co Kg Procédé de production d'une protéine recombinée à l'échelle industrielle
US7521240B2 (en) 2001-05-30 2009-04-21 Smithkline Beecham Corporation Chromosome-based platforms
WO2010135742A1 (fr) * 2009-05-22 2010-11-25 Merial Limited Plasmide sans antibiotique

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BALBAS P. ET AL.: "A pBRINT family of plasmids for integration of cloned DNA into the escherichia coli chromosome", GENE, vol. 172, 12 June 1996 (1996-06-12), pages 65 - 69, XP002935201 *
CHAUHAN S.S. ET AL.: "Construction of a new univerdal vector for insertional mutagenesis by homologous recombination", GENE, vol. 120, 21 October 1992 (1992-10-21), pages 281 - 285, XP002935203 *
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Cited By (15)

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Publication number Priority date Publication date Assignee Title
US7521240B2 (en) 2001-05-30 2009-04-21 Smithkline Beecham Corporation Chromosome-based platforms
US7160711B2 (en) 2001-08-06 2007-01-09 Degussa Ag Coryneform bacteria which produce chemical compounds I
WO2004069996A3 (fr) * 2003-02-05 2004-12-09 Degussa Bacteries et procede de production de composes chimiques au moyen desdites bacteries i
WO2006025702A1 (fr) * 2004-09-02 2006-03-09 Korea Advanced Institute Of Science And Technology Fragment d'adn lineaire pour deletion sans marqueur, nouvelle souche presentant une formation de biofilm inhibee et methode de preparation correspondante
US9683252B2 (en) 2007-05-17 2017-06-20 Boehringer Ingelheim Rcv Gmbh & Co Kg Method for producing a recombinant protein on a manufacturing scale
WO2008142028A1 (fr) * 2007-05-17 2008-11-27 Boehringer Ingelheim Rcv Gmbh & Co Kg Procédé de production d'une protéine recombinée à l'échelle industrielle
US10752930B2 (en) 2007-05-17 2020-08-25 Boehringer Ingelheim Rcv Gmbh & Co Kg Method for producing a recombinant protein on a manufacturing scale
AU2008252990B2 (en) * 2007-05-17 2015-01-15 Boehringer Ingelheim Rcv Gmbh & Co Kg Method for producing a recombinant protein on a manufacturing scale
EP3399042A1 (fr) * 2007-05-17 2018-11-07 Boehringer Ingelheim RCV GmbH & Co KG Procédé de production d'une protéine recombinante à l'échelle industrielle
CN102482679A (zh) * 2009-05-22 2012-05-30 梅里亚有限公司 无抗生素的质粒
EP3124613A1 (fr) * 2009-05-22 2017-02-01 Merial, Inc. Plasmide sans antibiotiques
US9217153B2 (en) 2009-05-22 2015-12-22 Merial, Inc. Antibiotic-free plasmids
RU2548809C2 (ru) * 2009-05-22 2015-04-20 Мериал Лимитед Плазмида без устойчивости к антибиотику
US10487334B2 (en) 2009-05-22 2019-11-26 Boehringer Ingelheim Animal Health USA Inc. Antibiotic-free plasmid
WO2010135742A1 (fr) * 2009-05-22 2010-11-25 Merial Limited Plasmide sans antibiotique

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