EP0912722A1 - Method of constructing vectors for homologous recombination directed mutagenesis - Google Patents
Method of constructing vectors for homologous recombination directed mutagenesisInfo
- Publication number
- EP0912722A1 EP0912722A1 EP98908603A EP98908603A EP0912722A1 EP 0912722 A1 EP0912722 A1 EP 0912722A1 EP 98908603 A EP98908603 A EP 98908603A EP 98908603 A EP98908603 A EP 98908603A EP 0912722 A1 EP0912722 A1 EP 0912722A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- vector
- sequences
- genomic dna
- selection cassette
- positive selection
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
-
- 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
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
Definitions
- the present invention relates to methods and vectors for making specific mutations in genes. More specifically, the invention relates to the use of a vector system useful in modifying the eukaryotic genome, particularly of embryonic stem cells, at precise and predefined loci by the means of homologous recombination.
- the vector usually contains a positive selection cassette which is flanked by the genetic information of the target locus to enrich for cells where the vector successfully recombines with the chromosomal DNA against the pool of non-recombinant cells.
- Stable integration leads to a long term resistance against certain pharmacological toxins. Examples are the resistance against G418, i.e., Geniticin, or Hygromycin by the action of the neomycin or hygromycin resistance genes, respectively.
- the position of the positive selection cassette in the chromosomal vector DNA can further lead to a mutation of the gene as in classical knockout experiments, i.e., inactivation of gene function.
- inactivation or modification of regulatory elements of the target gene as well as of domains of the transcribed/translated gene product could have positive, negative or modulatory effects on future target gene function.
- Homologous recombination that is carried out by the target DNA flanking the positive selection cassette, has to be selected against the background of unwanted non-homologous recombination that is thought to occur over the vector ends.
- a negative selection cassette positioned at the terminus of the vector will frequently be integrated by the non-homologous recombination events. Stable expression of the negative selection marker leads to cytotoxicity of otherwise non-cytotoxic agents.
- An example is the activated cytotoxicity of Gancyclovir by the action of the Herpes Simplex virus thymidine kinase gene product (HSV-TK) .
- HSV-TK Herpes Simplex virus thymidine kinase gene product
- the prior art is deficient in the lack of effective means of constructing vectors for homologous recombination directed mutagenesis. •
- the present invention fulfills this longstanding need and desire in the art.
- An object of the present invention is to simplify the construction of vectors for the targeted mutation of genes by homologous recombination in mammalian cells. These vectors facilitate the construction of specifically mutated cells, cell lines derived from the individually mutated cells, and cells for the use in the production of transgenic non-human animals.
- Another object of the present invention is to provide a vector system that avoids the drawbacks of conventional vector construction.
- the present invention provide a vector system and a new procedure that simplifies the cons tru ction of positive/negative selection cassettes. This new method reduces the time required for the construction of such vectors from 3-6 months to about 14 days.
- a particularly useful vector class contemplated by the present invention includes a linear lambda vector (lambdaKOS, i.e., knockout shuttle) for the construction of genomic DNA libraries that comprises: a stuffer fragment; an E. coli origin of replication; an antibiotic resistance gene; a yeast origin of replication; a selectable marker suitable for use in yeast; a negative selectable marker suitable for use in mammalian cells; LoxP sequences for Cre recombinase directed conversion of said linear lambda phage vector into an E. colilyeast shuttle plasmid.
- An additional vector contemplated by the present invention is a vector designed to specifically insert a positive selection cassette into cloned genomic DNA.
- the vector comprises an E.
- co li origin of replication an antibiotic resistance gene
- a selectable marker suitable for use in yeast a positive selectable marker suitable for use in mammalian cells
- unique restriction endonuclease sequences flanking the positive selectable marker so that the marker can be exchanged for another positive selection marker
- unique restriction endonuclease sequences for the excision of the positive selection cassette of the vector and restriction endonuclease sequences flanking the bacterial and yeast sequences to facilitate the removal of these sequences from the vector after yeast-mediated recombination into the genomic target site of the shuttle plasmid unique restriction endonuclease sequences flanking the bacterial and yeast sequences to facilitate the removal of these sequences from the vector after yeast-mediated recombination into the genomic target site of the shuttle plasmid.
- An additional embodiment of the invention provides a method of generating mutations at specific sites in cloned genomic DNA, comprising the steps of: cloning genomic DNA into a linear lambda vector; isolating a clone of interest; converting the linear lambda vector containing the genomic DNA of interest into a circular E. colilyeast shuttle vector; identifying genomic DNA sequences intended for targeting of the positive selection cassette; synthesizing deoxyoligonucleotides complementary to sequences flanking the site intended for targeting of the positive selection cassette to the circular genomic shuttle vector; attaching the deoxyolignucleotides to the positive selection cassette by ligation or PCR; co-transforming the E.
- colily ast shuttle vector containing the genomic DNA of interest and the modified positive selection cassette into a yeast host cell wherein an intact recombinant plasmid is selected for by culturing the yeast on an appropriate media by means of the gene products provided for by two independent yeast selectable markers of the E. co lilyQast shuttle vector and the positive selection cassette and is obtained by performing homologous DNA recombination between homologous regions of the vector containing genomic DNA and the synthetically derived sequences which had been ligated onto the ends of the positive selection cassette thereby generating a new recombinant vector with the positive selection cassette inserted into the genomic DNA sequences.
- colilye ⁇ st sequences of the positive selection cassette vector are removed, comprising the steps of: digesting the new recombinant vector with restriction endonuclease specific for the unique restriction endonuclease sequences incorporated into the positive selection cassette vector which flank the E. c ⁇ /tVyeast sequences in the vector; ligating said digested vector; and identifying ligation products lacking the E. colilyeast sequences desired to be removed.
- mutated animal cells consisting of: linearization of a new recombinant vector at a unique restriction endonuclease sequence outside the sequences of the cloned genomic DNA; introduction of linearized DNA into an animal cell; and selection of transduced cells that express a positive selectable marker.
- the animal cells are embryonic stem cells or any other cell type with the potential to generate an animal.
- a method for the production of non-human transgenic animal consisting of: introduction of mutated animal cells into animal embryos; placement of embryos containing mutated cells into the uterus of a female animal; and replacement of the nucleus of a fertilized egg with the nucleus containing the modified genomic DNA.
- genomic and sub-genomic libraries constructed in the linear lambda vector wherein the libraries are derived from genomic DNA isolated from the group consisting of animal cells, mammalian cells, rodent cells, murine cells and other higher eukaryotic cells.
- FIG. 1 shows a diagrammatic representation of a linear lambda vector (lambdaKOS) that is generally representative of the type of vector that may be used in the present invention.
- Figure 2 shows a diagrammatic representation of a E. coli/yeast shuttle vector that is generally representative of the type of vector that may be used in the present invention, and shows a general strategy for the method of insertion of the positive cassette into the genomic DNA sequence.
- Figure 3 shows an example of positive selection cassette.
- the complete URA/CAT-selection cassette can be removed by a digest with the endonuclease Sfil and replaced by any other desired positive selection cassette.
- FIG. 4 shows pMCS-1 constructed as a new plasmid, comprising: an inverted repeat of three endonuclease restriction sites (Sfil, Swal and AscI) flanking three unique endonuclease restriction sites (BamHI, EcoRI and Xhol) for the insertion of positive selection cassette. Any of the central three sites can be used to insert a selection cassette.
- insertional mutagenesis system shall refer to a genetic system which allows one to mutate a genomic locus by inserting a genetic marker with or without replacing genomic sequences at the site of insertion.
- targeted mutagenesis shall refer to mutation of a genetic locus by inserting or replacing parts of the locus with a selection cassette.
- the site of mutation is generally selected by homologous recombination.
- linear lambda vector shall refer to
- DNA prepared from lambda phages which is linear as compared to
- E. colilyeast shuttle vector shall refer to any plasmid which contains replication origins for plasmid survival, i.e., multiplication and selection in E. coli and yeast cells.
- Cre recombinase shall refer to the activity of the Cre recombinase protein, which recombines between short stretches of DNA comprising two LoxP elements.
- lambdaKOS genomic library shall refer to a collection of lambda phages which represent, by cumulative content of genomic DNA, either a total mammalian genome or a specified fraction thereof.
- negative selection vector shall refer to a vector containing a gene active in mammalian cells which allows the killing of such cells carrying the vector.
- Cre/LoxP recombination system shall refer to a system where the expression and activity of Cre- recombinase protein leads to a recombination effort between two cognate LoxP sequences.
- the present invention describes a method of DNA vector construction to substantially improve the engineering of targeted mutation of genes by homologous recombination in mammalian cells.
- the present invention provides an insertional mutagenesis system useful in constructing vectors for the targeted replication mutagenesis of mammalian cells by homologous recombination in which the mutagenesis system comprises: (a) a linear lambda vector for the cloning of genomic DNA to be flanked by negative selection markers; and (b) a vector for the insertion of a positive selection cassette into cloned genomic DNA.
- the particularly unique features of this methodology include the construction of genomic DNA libraries in vectors with negatively selectable markers flanking the cloning sites and replication origins for E. coli and yeast cells to insert a positive selection cassette into the cloned genomic DNA sequences by homologous recombination in yeast directed by synthetic DNA sequences ligated onto the ends of a yeast replication deficient positive selection cassette and identical to the targeted genomic DNA sequences.
- This methodology simplifies the construction of positive/negative selection cassettes and furthermore reduces the time required for the construction of such vectors from 3-6 months to about 14 days.
- a linear lambda vector (lambdaKOS) system was invented herein based on lambda phage cloning which allows the construction of representative genomic libraries of essentially every eukaryotic genome.
- This vector of the present invention comprises a stuffer fragment of DNA flanked by restriction endonuclease sequences to facilitate the replacement with and thereby the cloning of genomic DNA; an E. coli origin of replication; an antibiotic resistance gene; a yeast origin of replication; a selectable marker suitable for use in yeast; negative selectable markers suitable for use in mammalian cells; a direct repeat of recombinase sequences for recombinase directed conversion of the linear lambda phage vector into an E. colilyeast shuttle plasmid.
- this vector or any variants thereof are created by the use of different negative selectable markers.
- the recombinase sequence and corresponding recombinase are selected from the group consisting of LoxP sequences-Cre recombinase and Frt sequences-Flp recombinase or any similar substitute as would be readily known to one having ordinary skill in this art.
- An additional vector, i.e., the positive selection cassette vector, contemplated by the present invention is a vector designed to specifically insert a positive selection cassette into cloned genomic DNA.
- This vector comprises a plasmid origin of replication; an antibiotic resistance gene; a selectable marker suitable for use in yeast; a positive selectable marker suitable for use in mammalian cells ; unique restriction endonuclease sequences flanking the positive selectable marker so that the marker can be exchanged for another positive selection marker; a unique restriction endonuclease sequence for the linearization of the vector and restriction endonuclease sequences flanking the bacterial and yeast sequences to facilitate the removal of these sequences from the vector.
- the positive selection cassette vector or any variants thereof are created by the use of a different positive selection marker.
- An additional embodiment of the invention provides a method of generating mutations at specific sites in cloned genomic DNA, comprising the steps of: cloning genomic DNA into the linear lambda vector; isolating a clone of interest; converting the linear lambda vector containing the genomic DNA of interest into a circular E. coli/yeast shuttle vector by infecting a Cre recombinase expressing bacterial strain; identifying genomic DNA sequences intended for targeting of the positive selection cassette; synthesizing deoxyoligonucleotides complementary to sequences flanking the- site intended for targeting of the positive selection cassette to the circular E.
- E. coli/yeast shuttle vector attaching the synthetic deoxyoligonucleotides to the positive selection cassette by ligation or PCR (see Example 2); co-transforming said E . coli/y east shuttle vector containing the genomic DNA of interest and the modified positive selection cassette into a yeast host cell, wherein an intact recombinant plasmid is selected for by culturing yeast on an appropriate media by means of the gene products provided for by the yeast selectable markers of the E.
- the E. coli/yeas t shuttle vector and the positive selection cassette is obtained by performing homologous DNA recombination between homologous regions of the vector containing genomic DNA and the synthetically derived sequences which had been ligated onto the ends of the positive selection cassette thereby generating a new recombinant vector with the positive selection cassette inserted into the genomic DNA sequences.
- the E. coli/yeas t sequences of the positive selection cassette vector are removed, comprising the steps of: digesting the new recombinant vector with restriction endonuclease specific for the unique restriction endonuclease sequences incorporated into the positive selection cassette vector which flank the E. co li/yeast sequences in the vector; ligating the digested vector; and identifying ligation products lacking the E. coli/yeast sequences desired to be removed.
- the present invention is also directed to a method for the production of mutated animal cells consisting of: linearizing the recombinant vector at a unique restriction endonuclease sequence outside the sequences of the cloned genomic DNA; introduction of the linearized DNA into an animal cell; and selection of transduced cells that express the positive selectable marker.
- the animal cells are embryonic stem cells.
- the present invention is also directed to a method for the production of non-human transgenic animal consisting of: introduction of the mutated animal cells into animal embryos; placement of the embryos containing the mutated cells into the uterus of a female animal; and replacement of the nucleus of a fertilized egg with the nucleus containing the modified genomic DNA.
- the present invention is further directed to genomic and sub-genomic libraries constructed in the linear lambda vector wherein said libraries are derived from genomic DNA isolated from the group consisting of animal cells, mammalian cells, rodent cells, murine cells and other higher eukaryotic cells.
- the invention when compared to other techniques of homologous recombination vector construction offers the following advantages: First, the recombinant phage library has to be prepared only once for the particular genome of interest and can afterwards be amplified as a phage library in bacteria a million fold. Secondly, every gene locus is automatically ready for isolation with any gene probe of interest. Thirdly, the phage system allows the propagation of large chromosomal DNA which assists successful homologous recombination. Fourthly, a simple transfection of a Cre recombinase expressing bacterial strain creates a shuttle plasmid in which the genomic DNA is automatically flanked by negative selection cassettes. Fifthly, the shuttle plasmid not only allows the propagation in E.
- a "DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
- a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
- a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
- a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
- oligonucleotide or "deoxyoligonucleotide” as used herein is defined as a molecule comprised of two or more ribonucleotides or deoxyribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide.
- primer refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH.
- the primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, the source of primer and the method used.
- the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
- PCR refers to the polymerase chain reaction that is the subject of U.S. Patent Nos. 4,683,195 and 4,683,202 to Mullis, as well as other improvements known in the art.
- restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence .
- the labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to ultraviolet light, and others.
- a number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
- a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
- the linear lambda vector (lambdaKOS) has the following features from left to right (see Figure 1): cos end and left arm of the phage, synthetic LoxP fragment, HSV-tk gene, endonuclease recognition sites BamHI and Sail, stuffer fragment, endonuclease recognition sites Sail and BamHI, HSV-tk gene, plasmid vector (bacterial origin of replication, ⁇ -lactamase gene for ampicillin resistance), endonuclease recognition sites NotI, yeast origin of replication 2micron, yeast auxotrophic TRP1 gene, synthetic LoxP fragment (same relative orientation as the previous one) and the right arm of the phage and cos end.
- DNA prepared from the lytic growing phage can be digested by the endonucleases Sail and BamHI and the stuffer fragment replaced by genomic DNA partially digested by the endonuclease Sau3AI.
- a several fold representative genomic library of a eukaryotic genome can thereby be established. Every genomic fragment ( 10,000- 15,000 base pairs) is automatically flanked by a negative selection cassette on either site. Since the genomic library can be several fold redundant, virtually every genomic locus is represented by a lambdaKOS phage. Genomic clones of interest can be screened by classical filter hybridization of the lambda phage library.
- the insertion of the positive selection cassette (URA3/CAT-selection cassette) into the genomic DNA of pKOS is carried out by cotransfection of the cassette with the particular pKOS plasmid into yeast and selection for complementation of both auxothrophic requirements of the TRP/URA3-deficient yeast strain.
- the particular yeast strain used in this system requires the gene products of URA3 and TRP1 to survive and grow on selection plates.
- the TRP1 gene is delivered by the pKOS plasmid.
- the complementary URA3 gene is cotransfected as part of the replication deficient positive selection cassette.
- the URA3/CAT-selection cassette for cloning purposes is part of a high copy bacterial plasmid and has the following features (see Figure 3): endonuclease recognition sites Sfil, BamHI, Xhol, Swal and AscI, a yeast active URA3 gene, a bacterial active chloramphenicol acetyltransferase gene (CAT) gene and endonuclease - recognition sites Swal, AscI and Sfil.
- Any positive selection marker e.g. neomycin or hygromycin expression cassettes
- Digestion of the URA3/CAT-selection cassette with the endonuclease Sfil will generate incompatible overhangs (see Figure 2) to which synthetic double stranded oligonucleotides can be ligated.
- the sequences of the left and right oligonucleotides have to match the flanking sites of the desired integration site in the genomic DNA of the shuttle plasmid. Generally 40 base pairs of homology on both sites are sufficient for a successful integration of the positive selection cassette into the desired integration site of the pKOS plasmid.
- PCR polymerase chain reaction
- Primer A 40 base pairs of homology with the 5 ' flanking sequence of the target locus and 15-20 base pairs of the 5' end of the positive selection cassette.
- Primer B 40 base pairs of homology with the 3' flanking sequence of the target locus (opposite strand) and 15-20 base pairs of the 3' end of the positive selection cassette.
- the generated URA3/CAT-selection cassette amplicon can be contransfected into the yeast strain with the pKOS plasmid of interest using standard procedures.
- the yeast replication incompetent positive selection cassette After successful cotransfection of the yeast replication incompetent positive selection cassette with the replication competent E. coli/yeast shuttle plasmid (pKOS), only the yeast cells survive on -URA/-TRP selection plates where the positive selection cassette recombines with the shuttle plasmid.
- the plasmids are recovered from the yeast cells and transferred into E . c o l i which allows a further selection by plating on chloramphenicol and ampicillin containing plates using standard procedures.
- -Digestion of the plasmids with the endonuclease AscI or Swal and re-ligation eliminates the URA3/CAT part of the completed pKOS recombination vector which can be selectively grown in bacteria in ampicillin containing media.
- the complete URA/CAT-selection cassette can be removed by a digest with the endonuclease Sfil and replaced by any other desired positive selection cassette.
- pMCS- 1 which has the following features (see Figure 4): an inverted repeat of three endonuclease restriction sites (Sfil, Swal and AscI) flanking three unique endonuclease restriction sites (BamHI, EcoRI and Xhol) for the insertion of positive selection cassettes for later replacement of the URA3/CAT cassette from pKOS after yeast mediated recombination.
- any of the central three sites can be used to insert a selection cassette, e.g., a neomycin, and any of the flanking sites can be used to swap the selection cassette with the pKOS inserted URA3/CAT cassette using the respective endonuclease restriction sites.
- a selection cassette e.g., a neomycin
- flanking sites can be used to swap the selection cassette with the pKOS inserted URA3/CAT cassette using the respective endonuclease restriction sites.
- a lambdaKOS genomic library from the mouse inbred strain LEX- 1 has been successfully generated with a 10 fold representation of the mouse genome and used successfully several times to recover pKOS clones of the desired genomic loci (6 of 6 independent trials) for homologous vector construction.
- the planned vector construction was carried out using the above discussed procedures and all insertions of the URA3/CAT- selection cassette were proceeded without any improper recombination events in yeast which would generate useless pKOS constructs.
- HSV-TK HSV-thymidine kinase
- LambdaKOS genomic libraries or sublibraries are generally, but not exclusively, generated by cloning random fragments of genomic DNA between the phage arms. It is usually important to obtain sequence information from the inserted fragment ends in order to generate outside probes to analyze the mutated locus and to analyze and compare individual pKOS clones. Since the genomic insert is flanked on both sites by the HSV-TK cassettes, any pKOS sequencing primer oriented towards the insert would generate useless information.
- the construction of lambdaKOS allows the generation of two oligonucleotide primers which contain and reflect a single base pair different between the 5' end of the two HSV-TK cassettes: KOS-1 : 5'-accacactgctcgaggat
- KOS-2 5'-accacactgctcgacgga Both primers allow a direct sequencing of both genomic insert ends, respectively.
- the invented vector system consists of a completely new constructed lambda phage (lambdaKOS, i.e., knockout shuttle) which allows one to clone the complete genome of a eukaryotic organism in a negative selection vector to build a representative genomic library.
- lambdaKOS i.e., knockout shuttle
- Every target gene of that genome is accessible for any planned mutation after one single cloning step (i.e., the generation of the genomic library) which has to be carried out only once for an individual eukaryotic cell.
- the phage, containing the desired genomic locus can be isolated from the library which can easily be amplified and used for future clone isolation from other genomic loci.
- an E. coli/yeast shuttle plasmid (pKOS) can be obtained.
- the positive selection cassette is inserted into the plasmid by site-specific homologous recombination in yeast.
- the invention as compared to other techniques of homologous recombination vector construction has the following advantages: the recombinant phage library has to be prepared only once for the particular genome of interest and can afterwards be amplified as a phage library in bacteria a million fold. Automatically, every gene locus is ready for isolation with any gene probe of interest.
- the phage system allows the propagation of large chromosomal DNA which assists successful homologous recombination.
- a simple transfection of a Cre recombinase expressing bacterial strain creates a shuttle plasmid in which the genomic DNA is automatically flanked by negative selection cassettes.
- the shuttle plasmid not only allows the propagation in E. coli but also in yeast for which it can be selected for (TRP1) after successful transformation.
- the introduction of the positive selection cassette follows a published procedure (Nucleic Acid Res. 24: 4594-4596) but is further improved by the herewith described newly developed positive selection cassette and pMCS- 1 plasmid which simplify the construction of variants.
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WO2000055311A2 (en) * | 1999-03-17 | 2000-09-21 | E.I. Du Pont De Nemours And Company | Gene modification by homologous recombination |
US6548271B1 (en) | 1999-04-22 | 2003-04-15 | Lexicon Genetics Incorporated | Nucleic acids encoding human transporter proteins |
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US20020031829A1 (en) * | 2000-08-15 | 2002-03-14 | Brian Zambrowicz | Arrayed collection of genomic clones |
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US6376192B1 (en) | 2000-11-28 | 2002-04-23 | Baylor College Of Medicine | Method for screening of DNA libraries and generation of recombinant DNA constructs |
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Non-Patent Citations (5)
Title |
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NEHLS M ET AL: "Two large insert vectors, lambda PS and lambda KO, facilitate rapid mapping and targeted disruption of mammalian genes" BIOTECHNIQUES, vol. 17, no. 4, October 1994 (1994-10), pages 770-775, XP002179274 * |
See also references of WO9837175A1 * |
STORCK T ET AL: "Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse" NUCLEIC ACIDS RESEARCH., vol. 24, no. 22, 15 November 1996 (1996-11-15), pages 4594-4596, XP002076777 * |
TSUZUKI T ET AL: "Embryonic stem cell gene targeting using bacteriophage lambda vectors generated by phage-plasmid recombination" NUCLEIC ACIDS RESEARCH., vol. 26, no. 4, 15 February 1998 (1998-02-15), pages 988-993, XP002179275 * |
WATTLER S ET AL: "Construction of gene targeting vectors from lambda KOS genomic libraries" BIOTECHNIQUES, vol. 26, no. 6, June 1999 (1999-06), page 1150-1156, 1158, 1160 XP002179272 * |
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