JP2002514072A - Methods for construction of vectors for homologous recombination directing mutagenesis. - Google Patents

Abstract

  (57) [Summary] The present invention provides a new vector system for directing mutagenesis, and even a new method for simplifying the construction of recombinant vectors. The vector system homologous recombination techniques described above are used to modify the genome of eukaryotes, especially embryonic stem cells, at precise and predetermined loci. Moreover, it can be seen that the system can be used to generate new strategies for gene therapy and to generate genetically modified higher eukaryotes.

Description

DETAILED DESCRIPTION OF THE INVENTION Title of Invention Construction of vector for homologous recombination to direct mutagenesis Law                                Background of the InventionField of the invention   The present invention relates to a method and a vector for making a specific mutation of a gene. Further More specifically, the invention relates to a method for homologous recombination of eukaryotic genomes, particularly embryonic stem cells. Method for modifying genomes at precise and predetermined loci Related to the use of various vector systems.Description of related technology   Leading to chromosomal alteration and thereby modification of gene structure and / or expression It has been described in many different technologies. Against targeted mutagenesis These techniques are based on homologous recombination. Target mutation in the genome of a cell Genetically altered embryonic stem (E) with different general methodologies and specific genetic disorders S) The process of development of cell-derived mouse lines is well known (Bradly, 1991,Cur . Opin. Biotech., 2: 823-829).   A synthetic recombinant vector containing the genetic information of the target chromosomal locus is introduced into the cell. After that, recombine with genomic DNA. Vector is usually a pool of non-recombinant cells Targets enrich for cells whose vectors can be successfully recombined with chromosomal DNA It has a positive selection cassette adjacent to the genetic information of the locus. Cheap Fixed incorporation leads to long-term resistance to certain pharmacological poisons. Example is G41 8 ie neomycin against geneticin or hygromycin respectively Alternatively, it is resistance due to the action of the hygromycin resistance gene. Chromosome vector D The placement of the positive selection cassette in NA is a classic knockout experiment, Mutations in the gene for inactivation of the ability can be further derived. further Inactivation or modification of the regulatory elements of the target gene is dependent on the domain of the transcription / translation gene product. As well as the effects of positive, negative or regulating future target gene function Can have.   Homologous recombination is performed by target DNA adjacent to the positive selection cassette, Background unwanted heterologous recombination that may occur beyond the Must be selected as a password. Negative selection vectors located at the end of the vector Sets may be more frequently integrated by heterologous recombination results. Negative choice Stable expression of the label leads to the cytotoxicity of another non-cytotoxic drug. Example is a simple helper Gancyclo by the action of subviral thymidine kinase gene product (HSK-TK) Activation of vir cytotoxicity. Opportunities for homologous recombination are available on chromosomes Increase both the size of the vector DNA and the genomic DNA of the vector and target cells. It depends on the isogenecity between the vesicles.   Cloning and cloning of large chromosome fragments (5,000-15,000 base pairs) of the target gene This D in the terrier plasmid vector NA subcloning, mapping of gene structure, positive selection Seto integration, and finally, one of the homology vector arms with a negative selection marker Alternately, adjacency of both is a technically demanding task, typically during long builds Time (3-6 months) is required. Therefore, the construction of the recombinant vector itself It is most often the time limiting stage of a targeted mutagenesis experiment.   Construction of vectors for homologous recombination directing mutagenesis in previous techniques It is also imperfect, lacking effective methods. The present invention is based on this technology. It fulfills long-standing needs and desires.                                Summary of the Invention   It is an object of the present invention to target mutation of a gene by homologous recombination in a mammalian cell Is the simplification of the construction of vectors for These vectors have specific mutations Cells, cell lines derived from individual mutant cells, and transgenic non-human Facilitate the construction of cells used for the production of animal animals.   Another object of the invention is to provide a vector system that eliminates the disadvantages of conventional vector construction It is to be.   The present invention introduces a vector system and a new procedure to facilitate the construction of positive / negative selection cassettes. Supply. This new method reduces the time required to construct these vectors by 3-6. Months to about 14 days.   Particularly useful vectors contemplated by the present invention are stuffer Fragment, E. coli origin, antibiotic resistance gene, yeast origin of replication, Ys Selectable label suitable for use in cells, negative selectable label suitable for use in mammalian cells Labeled, linear lambda phage vector. coli / yeast shuttle plasmid Genomic DNA constructing the LoxP sequence for Cre recombinase that directs the transformation of DNA Linear lambda vectors for the construction of libraries (lambda KOS, Utoshuttle).   Additional vectors contemplated by the present invention provide a positive selection vector for the cloned genomic DNA. It is a vector designed to insert a set specifically. The vector Is the copy of E. E. coli origin, antibiotic resistance gene, selection suitable for use in yeast Label, positive selectable label suitable for use in mammalian cells, and another positive label Unique restriction end adjacent to the positive selectable sign to exchange for the selectable sign Nuclease, a unique restriction endonuclease for deletion of the vector positive selection cassette Yeast-mediated recombination of creatase and shuttle plasmids into genomic target sites. Bacterial and yeast sequences that facilitate removal of these sequences from the vector after Constructs adjacent restriction endonuclease sequences.   An additional embodiment of the invention is to mutate a specific site in cloned genomic DNA. Genomic DNA cloning in linear lambda vector, Isolation of target clone, circular E of linear lambda vector containing genomic DNA of interest . E. coli / yeast shuttle vector, intended to target positive selection cassette Genomic DNA sequence identification, positive selection cassette for circular genome shuttle vector Deoxyoligonucleotides complementary to sequences flanking the site intended for targeting Positive synthesis of deoxyoligonucleotides by tide synthesis, ligation reaction or PCR Attachment to selection cassette, E. coli containing genomic DNA of interest in yeast host cells. c Co-transformation of an oli / east shuttle vector and a modified positive selection cassette Constitutes a stage of recombination, in which the complete recombinant plasmid is E. coli. coli / yeast Choice of two independent yeasts, shuttle vector and positive selection cassette Selection by culturing yeast in a suitable medium according to the gene product supply method by labeling And inserted into the vector containing the genomic DNA and the resulting genomic DNA sequence. Positive selection cassette that generates a new recombinant vector with a positive selection cassette DNA assembly between the synthesized sequence and the homologous region attached to the end of the It is obtained by changing. Isolation of yeast plasmid, E. coli. E. coli transformation And E. After isolation of the E. coli plasmid, the positive selection cassette vector E. coli was isolated. coli / a And the new recombinant vector was replaced with E. coli in the vector. coli / Ys Unique restriction endonuclease integrated with the positive selection cassette vector adjacent to the Cleavage with a restriction endonuclease specific for the creatase sequence, E. connection and removal is required coli / yeast distribution Consists of the stage of identification of the ligation product lacking a sequence.   With unique restriction endonuclease sequences outside the sequence of the cloned genomic DNA Linearization of new recombinant vector, introduction of linear DNA into animal cells, positive selection Mutated animal cells that consist of the step of selecting transfected animal cells that express A method of production is also provided. Preferably, animal cells are embryonic stem cells or individual Any other cell type with somatic potential is good.   Yet another embodiment of the present invention is the introduction of mutant cells into animal embryos, maternal uterus. Transfer of embryos containing mutant cells to the nucleus containing the transformed genomic DNA Production of non-human transgenic animals consisting of stages of replacement of sperm nuclei Is the supply of methods for.   In addition, genomic and subgenomic libraries constructed with linear lambda vectors Where the library contains animal cells, mammalian cells, rodent cells, Genomic DN isolated from a group consisting of rat cells and other higher eukaryotic cells Derived from A.                             BRIEF DESCRIPTION OF THE FIGURES   The details of the features, advantages and objects of the above enumeration of the invention are as detailed as others that will be clear. To be able to obtain and understand the details, the invention briefly summarized above For a more detailed description of the embodiments with reference to the accompanying drawings, reference is made to certain embodiments. It is done by illuminating. These drawings form part of the specification. I do. However, the accompanying drawings illustrate selected embodiments of the invention, and It should be noted that the enclosure should not be considered as limiting.   FIG. 1 shows a linear lambda vector (LA) generally representing the type of vector used in the present invention. 1 represents a schematic depiction of a waste KOS).   FIG. 2 shows E. coli generally representing the types of vectors used in the present invention. coli / East shut And a method of inserting a positive cassette into genomic DNA. Represents a general strategy for   FIG. 3 shows an example of a positive selection cassette. Complete URA / CAT selection cassettes Can be removed by cleavage with nuclease SfiI, Can be replaced with a selection cassette.   FIG. 4 shows pMCS-1 constructed as a new plasmid, with a positive selection cassette. Three unique endonuclease restriction sites (BamHI, EcoRI and And XhoI) and three endonuclease restriction sites (SfiI, SwaI and AscI) In the reverse direction. All three central sites are selection cassettes Can be used for insertion.                             DETAILED DESCRIPTION OF THE INVENTION   Here, "insertion mutagenesis system" refers to the genome sequence at the insertion site. Mutation at the locus by insertion of genetic markers with or without Genetics that allow rubbing Used as a reference to the system.   Here, “targeted mutagenesis” refers to the insertion of a locus by a selection cassette. Or used as a reference to mutation of a locus by a replacement moiety. suddenly The site of mutation is generally selected by homologous recombination.   Here, “linear lambda vector” is infected with lambda phage, and Lambda phage Cre expression in which phage DNA binds to circular plasmid DNA It was prepared from lambda phage which was linear in contrast to DNA prepared from bacteria. Used as a reference to prepared DNA.   Here, “E. coli / east shuttle vector” refers to E. coli. coli and Ys Survive in the cell, including the origin of replication of the propagated and selected plasmid. Used as a reference to any plasmid.   As used herein, “Cre recombinase” refers to the short DNA of two LoxP elements. With the activity of the Cre recombinase protein to be recombined between It is used as   Here, “lambda KOS genomic library” refers to the genomic DNA accumulation. Depending on the context, either the entire mammalian genome or instead specific parts Used as a reference to the lambda phage assembly.   As used herein, "negative selection vector" refers to a vector that has a vector in mammalian cells. Mentions vectors with gene activity that allow these cells to be killed, It is used as   Here, "Cre / LoxP recombination system" means Cre recombinase protein A system in which the expression and activity of E. coli leads to a recombination effect between two cognate LoxP sequences. Used as mentioned.   The present invention is directed to the work of targeted mutation of genes by homologous recombination in mammalian cells. A method for constructing a DNA vector that sufficiently improves the above is described. To embody one, The present invention relates to vectors for targeted replication mutagenesis of mammalian cells by homologous recombination. -Provide an efficient insertion mutagenesis system for construction. Mutagenesis there The system is based on (a) the cloning of genomic DNA flanked by negative selection markers. A linear lambda vector for (a) and (b) a negative selection cassette A vector for insertion into A is constructed.   A very unique feature of this methodology is the cloning site and E. coli. coli and Ys For positive cells, the positive selection cassette is inserted into the yeast cloning genomic DNA sequence. And binds to the end of the positive selection cassette lacking yeast replication and Insertion replication by homologous recombination indicated by a synthetic DNA sequence identical to the sequence Genomic DNA construction in vectors with negative selectable markers adjacent to the origin It is to include. This methodology simplifies the construction of positive / negative selection cassettes and furthermore Reduces the time required to construct these vectors from 3-6 months to about 14 days .   The linear lambda vector (lambda KOS) system is essentially Lambda phage allows construction of a typical genomic library of all eukaryotic genomes It was invented on the basis of cloning. This vector of the present invention is genomic DNA, replicated E. coli origin, antibiotic resistance gene, yeast origin of replication, use in yeast Selectable labels suitable for use with cells, negative selectable labels suitable for use with mammalian cells, lines From a lambda phage vector. conversion to E. coli / yeast shuttle plasmid Linear repeat substitution of the recombinase sequence for the indicated recombinase and Furthermore, the DNA flanking the restriction endonuclease sequence to facilitate cloning Construct the stuffer fragment of A. If possible, this vector Or alternative mutants are created by using another negative selectable marker Better. In this insertional mutagenesis system, the recombinant enzyme sequence and Recombinases are LoxP sequence-Cre recombinase and Frt sequence-Flp recombinase Or a similar substitute that can be easily understood to have normal skills by this technique? Selected from the group consisting of   Additional vectors contemplated by the present invention, namely positive selection cassette vectors -Will insert the positive selection cassette specifically into the cloned genomic DNA. Vector. This vector is a plasmid origin of replication, antibiotic resistance inheritance Selectable label suitable for use in yeast, yeast, positive label suitable for use in mammalian cells Selectable sign, the sign can be exchanged for another positive select sign, Adjacent to a selectable sign Unique restriction endonuclease sequences that facilitate removal of sequences from vectors Direct restriction endonuclease sequences and vector flanking bacterial and yeast sequences Consists of unique restriction endonuclease sequences for linearization. If possible, Positive selection cassette vectors or alternative variants should use a different positive selection marker. It is better to be created by using   Additional embodiments of the invention are directed to mutations at specific sites in cloned genomic DNA. Supply of methods to generate differences, including cloning in linear lambda vectors, Isolation of heart target clones, linear lambda vector containing genomic DNA of interest Was infected with a bacterial strain expressing Cre recombinase to form cyclic E. coli. coli / East shuttle vector The genomic DNA sequence intended to target the positive selection cassette, Annular E. Intended for targeting positive selection cassettes to E. coli / yeast shuttle vectors Synthesis and ligation of deoxyoligonucleotides complementary to the sequence adjacent to the site Alternatively, PCR of synthetic deoxyoligonucleotides into a positive selection cassette by PCR. Attachment (see Example 2), containing genomic DNA of interest in yeast host cells E. coli / yeast shuttle vector and modified positive selection cassette simultaneously Transformation, in which the complete recombinant plasmid is E. coli. coli / yeast shuttle Vectors and remains provided by the selectable marker for yeast in the positive selection cassette Selected by yeast culture in an appropriate medium by the gene product approach, A-containing vector homology region and There a new set with a positive selection cassette inserted into the genomic DNA sequence Synthetically linked to the end of the positive selection cassette that generates the replacement vector. Comprising the steps obtained by performing homologous DNA recombination between the generated sequences. You. Preferably, the positive selection cassette E. coli / yeast sequences should be removed Is often used in the vector. positive selection cassette flanked by E. coli / yeast sequences Specific restriction endonuclease sequences incorporating the vector Cleavage of a new recombinant vector by nuclease, ligation of the cleavage vector, E. which is desired to be removed confirmation of the ligation product lacking the E. coli / yeast sequence Be composed.   The present invention also dictates a method for the production of a mutant animal, which comprises Line of the recombinant vector with unique restriction endonuclease sequences outside the sequence of A And selecting the transfected cells that express the positive selectable label. Preferably, the animal cells are embryonic stem cells.   The invention also dictates a method of producing a non-human transgenic animal, which Introduction of mutant animal cells into animal embryos, transfer of embryos containing mutant cells to maternal uterus It consists of planting and replacement of a nucleus containing modified genomic DNA with a fertilized egg nucleus.   The invention further relates to genomic and subgenomic libraries constructed with linear lambda vectors. Command library, wherein the library comprises animal cells, mammalian cells, Isolated from the group consisting of rodent cells, murine cells, and other higher eukaryotic cells From genomic DNA obtained.   When the invention is compared with other techniques for construction of homologous recombination vectors, the following advantages are obtained. I can do it. First, the recombinant phage library can be used for individual genomes of interest. It can be prepared only once, and then used as a phage library in bacteria. Can be expanded 1000 times. Second, each locus is the genetics of interest. The child probe automatically prepares for separation. Third, the phage system Large chromosomal DNA can be propagated to help successful homologous recombination. No. Fourth, simple transfection of Cre recombinant enzyme expressing bacterial strains A Automatically creates a shuttle plasmid adjacent to the negative selection cassette. Fifth , The introduction of the positive selection cassette was performed according to published methods (Nuclec Acid Res , 24: 4594-4596), but the newly developed positive selection case described here. It is further improved by pMCS-1 which simplifies construction of mutants and mutants.   As in the present invention, these are within the skill of the art, , Immunological and recombinant DNA techniques. These techniques are sentence It is fully represented by the offering. For example, Maniatis, Fritsch & Sambrook “Mole cular Cloning: A Laboratory Manual (1982); “DNA Clonig: A Practical Appr oach ”Volumes I and II ((DN Glover ed. 1985);“ Oligonucleotide Synt hesis "(MJ Gait ed. 1984);" Nucleic Acid Hybridization "[BD Hames & S. J. Higgis eds. (1985)]; “Transcriotion and Translation” [B. D. Hames & S. J. Higgins eds. (1984)]; “Animal Cell Culture” [R. I. Fre shery, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986) ]; See Perbal, “A Practical Guide To Molecular Cloning” (1984) .   The following terms are defined to have the following definitions: "DNA molecule" Is a deoxyribonucleotide in either a single-stranded or double-stranded helix Used for polymers of (adenine, guanine, thymine, or cytosine) . The term is used only in the context of the primary and secondary structure of the molecule, but it is a special tertiary structure This is not a limitation. Thus, this term is especially useful for linear D Found on NA molecules (eg, restriction fragments), viruses, plasmids and chromosomes Including double-stranded DNA. In this discussion, the untranscribed strand of DNA will be It is performed on the structure for the array in a direction along the 5 ′ to 3 ′ direction (ie, , The strand has a sequence homologous to the mRNA).   DNA "decoding sequences" are transcribed in vivo under the control of appropriate regulatory sequences. A double-stranded DNA that is translated into a polypeptide. The boundary of the decoding sequence is 5 '(amino ) Determined by the start codon at the end and the translation stop codon at the 3 '(carboxyl) end Is done. Decoding sequences include eukaryotic sequences, cDNA from eukaryotic mRNA, eukaryotic Genes derived from biological (eg, mammalian) DNA Includes, but is not limited to, nome DNA and even synthetic DNA sequences. No. The polyadenylation signal and transcription termination sequence are usually 3 'to the decoding sequence. Will be located.   As used herein, "oligonucleotide" or "deoxyoligonucleotide" The term "nucleotide" refers to two or more, generally three or more oligonucleotides Alternatively, it is defined as a molecule composed of a deoxyoligonucleotide. That Exact size depends on many factors that depend on the ultimate function and use of the oligonucleotide. Will depend on   As used herein, the term “primer” refers to a primer as a purified restriction fragment. Complementary to the nucleic acid strand, whether a potential product or a synthetic product. Under conditions where primer extension synthesis occurs, ie, nucleotide and DNA Synthesized when an inducing reagent such as merase is present and under the appropriate temperature and pH conditions Referred to as an oligonucleotide that can act as a point of initiation . Primers are either single-stranded or double-stranded and required in the presence of an inducing reagent. The synthesis of the desired extension product must be started for a sufficiently long time. Primer accuracy The exact length depends on a variety of factors, including temperature, primer material, and method used. Is determined. For example, for diagnostic applications, depending on the complexity of the target sequence, Can a lignonucleotide primer contain only a few nucleotides? Maybe, but typical contains 15-25 or more nucleotides .   As used herein, the term "PCR" refers to other improvements known in the art. Added to Millis and U.S. S. Patent Nos. 4,689,195 and 4,683,202 It mentions the polymerase chain reaction.   The terms "restriction endonuclease" and "restriction enzyme" as used herein Will form double-stranded DNA at or near the specific nucleotide It refers to bacterial enzymes that cleave.   The most commonly used labels in these studies include radioactive elements, enzymes, and ultraviolet light. Chemicals that fluoresce when irradiated, and others are used. Many fireflies Light substances are known and can be used as labels. These include, for example, Fluorescein, rhodamine, auramine, texa red, AMCA blue, And Lucifera Yellow. Special detectable substances are prepared in the body of the goat Anti-rabbit antibody linked to fluorescein by isothiocyanate.   The following examples are provided for the purpose of illustrating various embodiments of the invention, and This does not mean that the present invention is limited.                                 Example 1 Creation of linear lambda vector (lambda KOS)   The vector system is a typical genomic library for essentially all eukaryotic genomes. It was created on the basis of lambda phage cloning that allows the construction of Lee. Linear Lambda Vector (La (Muda KOS) has the following characteristic parts from left to right (see FIG. 1). cos end and Left arm of phage, synthetic LoxP fragment, HSV-tk gene, endonuclease recognition unit Position BamHI and SalI, stuffer fragment, endonuclease recognition site SalI and Bam HI, HSV-tk gene, plasmid vector (bacterial origin of replication, ampicillin resistance Β-lactamase gene for sex), endonuclease recognition site NotI, 2 mi Yeast origin of clone replication, yeast auxotrophic mutant TRP1 gene, synthetic LoxP fragment Pieces (in the same direction relative to the previous one) and the right arm and cos end of the phage. Lysis DNA prepared from the elongated phage contains endonucleases SalI and BamHI, and Replaced by genomic DNA partially cut by endonuclease Sau3AI Can be cut with the stuffer fragment. Several times the size of eukaryotic genomes A genomic library can thereby be established. Each genomic fragment (10,0 (00-15,000 base pairs) automatically flanks the negative selection cassette at any site. Because genomic libraries are several fold in excess, virtually every genomic library is The locus is represented by lambda KOS phage. Genome loans of interest are Sieving by classical filter hybridisation of the muda phage library Can be divided. Bacterial strain expressing Cre recombinase with selected phage Infection is advanced copy E. coli / yeast shuttle plasmid (pKOS) Allows automatic Cre-mediated recombination of muda phage.                                 Example 2Insertion of positive selection cassette (URA3 / CAT-selection cassette)   pKOS positive selection cassette for genomic DNA (URA3 / CAT-selection cassette) Insertion) is for simultaneous tracing of the cassette with the unique pKOS plasmid in yeast. Auxiliary Mutations in Both Nussfection and TRP / URA3-Deficient Yeast Strains Implemented by selection for complementarity of requirements. Specials used in this system New yeast strains are used for URA3 and TRP1 for survival and growth on selection plates. Request a gene product. The TRP1 gene is carried by the pKOS plasmid. The complementary URA3 gene was co-transfected as part of a replication lacking the positive selection cassette. Receive an action.   URA3 / CAT-selection cassettes for cloning purposes are high copy bacteria It is a part of a plasmid and has the following features (see FIG. 3). Endonuclease Recognition sites SfiI, BamHI, XhoI, SwaI and AscI, yeast active URA3 gene, Bacterial gloramphenicolol acetyltransferase (CAT), and endonuclease Rease recognition sites SwaI, AscI, and SfiI. Any positive selection sign (eg Neo Mycin or hygromycin expression cassette) Before the endonuclease recognition site BamHI of the URA3 / CAT-selection cassette. And XhoI.   Endonuclease S of URA3 / CAT-selection cassette Incompatibility with fiI cleavage can link synthetic duplex oligonucleotides A protrusion can be generated (see FIG. 2). Sequence of left and right oligonucleotides Does not match the site adjacent to the desired integration site in the genomic DNA of the shuttle plasmid. I have to. Generally positive if 40 base pairs of homology at both sites Enough for successful integration of the cassette into the desired integration site of the pKOS plasmid It is. The very simple task of attaching 40 base pairs of homology is as follows from 5 'to 3' Polymerase Chain Reaction Using Oligonucleotide Primers (PCR). Primer A: 5 ′ flanking sequence of target locus and positive 40 base pairs homology with 15-20 base pairs at the 5 'end of the selection cassette. Primer -B: 3 'flanking sequence (opposite side chain) of target locus and 3' end of positive selection cassette 40 base pairs homology with 15-20 base pairs of   The generated URA3 / CAT-selection cassette amplicon was generated using standard techniques. Co-transfection of yeast strain with pKOS plasmid of interest Can be                                 Example 3Further selection of homologous recombination vectors   Complete E. of replication. Es with E. coli / Yeast Shuttle Plasmid (pKOS) Simultaneous transfer performed without the problem of incomplete positive selection cassettes After the injection, only the yeast cells have a positive selection cassette paired with the shuttle plasmid. Survive with a change -URA / -TRP selection plate. Step Rasmid is recovered from yeast cells and chloramphenicol and Making more choices than plating on plates containing ampicillin E. transferred to E. coli. Plasmids are replaced with endonuclease AscI or Swa Cleavage and religation by I selectively grow in bacteria on ampicillin-containing media To remove the URA3 / CAT portion of the complete pKOS recombinant vector Leave. Alternatively, the complete USR / CAT-selection cassette is an endonucleated Digestion with SfiI and replacement with another desired positive selection cassette. You can leave.   To improve this replacement procedure, a new plasmid pMCS with the following features: -1 was constructed (see FIG. 4). UR from pKOS after yeast-mediated recombination 3 for the positive selection cassette insertion for later replacement of the A3 / CAT cassette. Adjacent to two unique endonuclease restriction sites (BamHI, EcoRI, and XhoI) Reverse repeats of three endonuclease restriction sites (SfiI, SwaI, and AscI) . All three central sites are for insertion of a selection cassette, eg, neomycin. Can be used for each of the selected cassettes Insert the URA3 / CAT cassette using these endonuclease restriction sites. Can be used to replace the pKOS. Finally, by homologous recombination Complete vectors for transfer to mammalian cells for genes that target animal cells Before infection Linearize using endonuclease NotI.                                 Example 4Generation of Lambda KOS Genome Library   Lambda KOS genomic library from mouse inbred strain LEX-1 Occurs up to 10 times the genome without any problems, and hopes for construction of homologous vectors Recovery of pKOS clones at different genomic loci (six in six separate tests) ) Was used without problems. The planned vector structure in all cases Construction is performed according to the method discussed above and all URA3 / CAT selection cassettes Insertion of any undesired recombination that results in unnecessary pKOS constructs Executed without consequences. Incorporation of positive selection cassette into pKOS The precise recombination performed by the yeast system is essentially that pKOS Two identical HSV-thymidine kinases are perfect substrates for the recombination process (HSV-TK) Transport is surprising. Two HSV-TK cassettes Since the kits are built in the opposite direction in pKOS, the two cassettes Recombination occurring at an intermediate point causes inversion of the intervening DNA, but is lacking Or no other deleterious effect on the pKOS plasmid.   Lambda KOS genomic libraries or sublibraries are generally, but not exclusively, Not exclusive, for cloning any fragment of genomic DNA between phage arms Be produced more. Analysis of mutant loci and individual pKOS clones, Obtain sequence information of the end of the insert to generate an outer probe for comparison That is generally important. Genomic insertions flanked on both sides of the HKS-TK cassette Therefore, pKOS sequence primers oriented for insertion are useful information Will not produce. The construction of lambda KOS involves two HSV-TK cassettes Two oligonucleotides that have and reflect only one base pair difference between the 5 'ends of the Tide primers can be generated.   Both primers are used for direct sequencing of both genomic inserts Can be done.                                 Example 5   The vector system of the invention provides a negative for constructing a typical genomic library. Cloning a complete genome of eukaryotic organic matter in a selection vector Completely newly constructed lambda phage (lambda KOS, knock- Uto Shuttle). Each target genetic of this means of that genome Offspring only needs to be performed once for each eukaryotic cell One cloning Any planned abrupt changes after a stage (ie, the creation of a genomic library) Can be used for different types. Phage containing the genomic locus of interest can be easily expanded. Can be used for future clone isolation from other genomic loci. A library that can be Lee can be separated. Self-mediated by Cre / LoxP recombination system After dynamic subcloning, E. coll / yeast shuttle plasmid (pKOS ) Can be obtained. Positive selection cassette is site-specific homologous recombination in yeast Into the plasmid.   The invention has the following advantages as compared to other techniques for the construction of homologous recombination vectors. Recombinant phage library prepared once for each individual genome of interest And then increase 1000-fold as a phage library in bacteria. Can be wide. Automatically, each locus is a gene profile of interest. Prepares for separation. Phage system helps troublesome homologous recombination Large chromosomal DNA can be propagated. Cre recombinant enzyme expression Simple transfection of the strain automatically reduces the genomic DNA Create a shuttle plasmid adjacent to the kit. The shuttle plasmid is E. coli. col Yeast that can be selected after successful transformation as well as within i They can proliferate even if they are. The introduction of the positive selection cassette is a published method ( Nucleic Acid Res. 24: 4594-4596) but follow the new rules described here. PMCS-1 plasmid that simplifies construction of positive selection cassettes and mutants developed in It is further improved by Sumid.   The patents or publications mentioned in this specification are not incorporated herein by reference. Show the level of their special skills. These features Permission and publication are each individually embodied by reference specifically and individually As such, it is embodied here to a similar extent.   One special skill of this technology is that it accomplishes its purpose, That they are well adapted to what they are inherent in Will evaluate easily. Methods, procedures, treatments and specificizations described here This implementation with the compound is a current representative of the selected embodiment and is a typical example. It is not intended to limit the scope of the invention. Changes therein and other uses Within the scope of the invention as defined in the claims. It will happen to their special skills.

────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Watler, Sigrid             United States 77381 Texas, The             Woodland, Holly Creek Co             G 333, epi. 602

Claims (1)

[Claims] 1. Construction vector for targeted mutagenesis of mammalian cells by homologous recombination Effective insertion mutagenesis system. (A) Linear lambda for cloning genomic DNA adjacent to a negative selection marker Vectors, and (B) Vector for insertion of positive selection cassette into cloned genomic DNA   The mutagenesis system described above. 2. The insertion mutagenesis system of claim 1 (A) Restriction endonuclease sequences that facilitate cloning of genomic DNA Adjacent DNA stuffer fragment (B) E. copy. coli origin (C) Antibiotic resistance gene (D) East origin of duplication (E) Selectable labels suitable for use in yeast (F) a negative selectable marker, located 5 'to said negative selectable marker; A position 3 'to the operable promoter element and the negative selectable marker. Labels containing a rear denier site (G) E. of linear lambda phage vector into E. coli / yeast shuttle plasmid Direct repetition of a recombinase sequence for a recombinase directing conversion   The linear lambda vector comprising: 3. The insertion mutagenesis system of claim 2, wherein the LoxP sequence-Cre recombination. Said recombination selected from the group consisting of: Enzyme sequences and corresponding recombinant enzymes. 4. The insertion mutagenesis system of claim 1 (A) E. of the copy. coli origin (B) Antibiotic resistance gene (C) Selectable labels suitable for use in yeast (D) a positive selectable marker, located 5 'to said positive selectable marker; The position 3 'to the operable promoter element and the positive selectable marker described above. Labels containing rear denylation sites (E) a positive selectable mark so that the sign can be exchanged for another positive selectable sign; Unique restriction endonuclease sequences flanking insights (F) positive selection prior to ligation of the synthetic deoxynucleotide to the end of the cassette Unique restriction endonuclease sequence for removal of cassettes (G) adjacent to bacterial and yeast sequences that facilitate removal of those sequences from the vector. Flanking restriction endonuclease sequences Vector for insertion of a positive selection cassette consisting of 5. Mutagenesis at specific sites in cloned genomic DNA How to generate (A) genomic DNA cloning in the linear lambda vector of claim 1 (B) Isolation of clones of interest (C) Linear line containing genomic DNA of interest due to infection of Cre recombinant enzyme expressing bacterial strain The circular E. lambda vector. Conversion to E. coli / Yeast Shuttle Vector (D) Identification of genomic DNA intended as a target for the positive selection cassette (E) The cyclic E. positive selection cassette for E. coli / yeast shuttle vector Of Deoxyribonucleotides Complementary to Sequences Flanking Sites of Intended Target (F) positive reaction of the synthetic deoxyribonucleotide by ligation reaction or PCR; Selection cassette attachment (G) the E. coli containing the genomic DNA of interest in yeast host cells. coli / Co-transformation of yeast shuttle vector and modified positive selection cassette, where Completely recombinant plasmids provide a gene product supply with a yeast selectable marker. Method, the complementary region of the vector containing genomic DNA and the New recombinant vector with a positive selection cassette inserted into the DNA sequence Homology D between synthetically derived DNA linked to the end of the positive selection cassette to be generated Obtained by causing NA recombination.   It consists of stages. 6. A method for generating a mutation at a specific site of the cloned genomic DNA according to claim 5, There the positive selection cassette E. coli / yeast sequences are removed. 7. The method of claim 6, wherein said removing is performed. (A) the recombinant vector having a positive selection cassette inserted into a genomic DNA sequence; E. of said vector in a positive selection cassette adjacent to the E. coli / yeast sequence. Restriction endonuclease specific for the inserted unique restriction endonuclease sequence Cutting with Aase (B) ligation of the cleavage vector, and (C) it is desired to remove of ligation products lacking E. coli / yeast sequences   It is composed of 8. Methods for producing mutant animal cells (A) unique restriction endonuclease sequence outside the sequence of the cloned genomic DNA The recombinant vector having a positive selection cassette inserted into the genomic DNA sequence in Linearization (B) introduction of the linear DNA into animal cells (C) Selection of transfected cells expressing the positive selection marker (D) Selection of transfected cells expressing the positive selection label and not expressing the negative label   It consists of stages. 9. The method of claim 9, wherein said animal cells are embryonic stem cells or ontogeny. Other cell types with potential. 10. Methods of non-human transgenic animal production, (A) Injecting the mutant animal cell of claim 8 into an animal embryo (B) transplantation of an embryo containing the mutant cell into a maternal uterus (C) Replacement of fertilized egg nucleus with nucleus containing modified genomic DNA     It is composed of