Method for genetic modification of target endogenic gene or chromosomal locus (variants) and uses thereof

FIELD: gene engineering.

SUBSTANCE: invention relates to method for modification target endogenic gene or chromosomal locus in eucaryotic cells. Claimed method includes production of large cloned genomic fragment having more than 20000 n.p. and designing based on the same large targeting vector (LTVEC) by using bacterial homological recombination. Further LTVEC is introduced into eucaryotic cells to modify endogenic gene or chromosomal locus. Finally assay is carried out to determine of allele modification in such cells. Also disclosed is application said cells for generation of organisms carrying such genetic modification.

EFFECT: method for modification with large DNA sequences.

26 cl, 6 dwg, 2 tbl, 5 ex

 

This application claims priority to Patent application U.S. No. 09/732234, filed December 7, 2000, and Provisional application U.S. No. 60/244665, filed October 31, 2000. Throughout this application refer to various publications. Descriptions of these publications in their entirety are included thereby by reference in this application.

The scope of the invention

The scope of the present invention is a method of construction and use of large DNA vectors for targeting through homologous recombination, the endogenous genes and chromosomal loci in eukaryotic cells and modification of these genes and loci in any desired manner. These were aimed at large DNA vectors for eukaryotic cells, called LTVEC, derived from fragments of the cloned genomic DNA, larger than the fragments that are commonly used in other approaches for performing homologous targeting in eukaryotic cells. In addition, the scope of the present invention provides a fast and convenient way to detect eukaryotic cells in which this LTVEC was correctly aimed, and modified desired endogenous gene (genes) or chromosomal locus (loci). This area also includes the use of these cells to generate organisms that carry this genetic modification, these organisms and methods of the x application.

Introduction

The use of LTVEC provides significant advantages over existing methods. For example, because they are derived from DNA fragments larger than fragments, currently used to generate targeting vectors, LTVEC can be more quickly and more conveniently generated from available libraries of large genomic DNA fragments (such as the library YOU library of bacterial artificial chromosome library and RACES)than the target vectors obtained using currently available technologies. Additionally, there may be generated a more convenient way than using current technology, large modifications, and variations, covering large areas of the genome.

In addition, this invention takes advantage of long regions of homology to increase the frequency of targeting "hard to target" loci, and also reduces the benefit, if it exists at all, the use of isogenic DNA in these target vectors.

Thus, this invention provides a fast, convenient and improved way for system modifications virtually all endogenous genes and chromosomal loci specific organism.

Background of the invention

It was shown that n is zalivanje genes through homologous recombination between homologous exogenous DNA and the endogenous chromosomal sequences is extremely valuable by creating deletions, indels designed mutations, accurate gene mutations, introduction of transgenes or other genetic modifications in mice. Current methods include the use of standard target vectors, and their regions of homology relative to endogenous DNA typically have an overall length of less than 10-20 TPN, for introduction of the desired genetic modification in mouse embryonic stem (ES) cells with the subsequent injection of these modified ES cells into mouse embryos to transfer these constructed genetic modifications into the mouse germ line (Smithies et al., Nature, 317:230-234, 1985; Thomas et al., Cell, 51:503-512, 1987; Koller et al., Proc Natl Acad Sci USA, 86:8927-8931, 1989; Kuhn et al., Science, 254:707-710, 1991; Thomas et al., Nature, 346:847-850, 1990; Schwartzberg et al., Science 246:799-803, 1989; Doetschman et al., Nature, 330:576-578, 1987; Thomson et al., Cell 5:313-321, 1989; DeChiara et al., Nature, 345:78-80, 1990; U.S. Patent No. 5789215, issued August 4, 1998 in the name of GenPhann International). In these existing methods, the detection of rare ES cells, in which the standard target vectors were correctly targeted and modified desired endogenous gene (genes) or chromosomal locus (loci), requires information sequence outside of the homologous target sequences contained in this targeting vector. Tests for successful targeting include standard blotting on the Southern or PR the term PCR (Cheng, et al., Nature, 369:684-5, 1994; Foord and Rose, PCR Methods Appl, 3:S149-61, 1994; Ponce and Micol, Nucleic Acids Res, 20:623, 1992; U.S. Patent No. 5436149 issued to Takara Shuzo Co., Ltd.) from sequences outside of the targeting vector and stretching all the shoulder homology (see Definitions); thus, due to considerations of size, which restrict these methods, the dimensions of the shoulders homology limited to sizes smaller than 10-20 TPN, in General (Joyner, The Practical Approach Series, 293, 1999).

Extremely useful would be the ability to use the target vectors with shoulders homology, greater than used in existing methods. For example, such target vectors could be faster and more convenient way to generate from the available libraries containing large genomic inserts (e.g. libraries YOU or RACES)than the target vectors obtained using currently available technologies, such genomic inserts must be extensively characterized and described in the order ("pruned") before use. In addition, large modifications, and variations, including large genomic regions, could be more easily generated and with fewer stages than when using current technologies. In addition, the use of long regions of homology could increase the frequency at which celivanja on "hard to target" loci in eukaryotic cells, because targeting homologous recombination in eukaryotic cells is likely to be associated with overall homology contained in the targeting vector (Deng and Capecchi, Mol Cell Biol, 12:3365-71, 1992). In addition, increased frequency of the target obtained using long shoulder homology, would reduce any potential benefits that can be obtained from the use of isogenic DNA in these target vectors.

The problem of constructing exact modifications in very large genomic fragments, such as fragments, cloned libraries YOU, was largely solved by using homologous recombination in bacteria (Zhang, et al., Nat Genet, 20:123-8, 1998; Yang et al., Nat Biotechnol, 15:859-65, 1997; Angrand, et al., Nucleic Acids Res, 27:e16, 1999; Muyrers et al., Nucleic Acids Res, 27:1555-7, 1999; Narayanan, et al., Gene Ther, 6:442-7, 1999), allowing the construction of vectors containing large regions of homology relative to the endogenous eukaryotic genes or chromosomal loci. However, after they are constructed, these vectors usually were not applicable for the modification of endogenous genes or chromosomal loci by homologous recombination because of the difficulty of correct detection of rare events targeting, when the shoulders of homology are larger than 10-20 TPN (Joyner, The Practical Approach Series, 293, 1999). Thus, vectors, GE is Araruama using bacterial homologous recombination of the genomic fragments YOU must be still in order ("pruned") for use as targeting vectors (Hill et al., Genomics, 64:111-3, 2000). Thus, there is still a need for rapid and convenient methodology, which provides the possibility of using targeting vectors containing large regions of homology, thus, to modify endogenous genes or chromosomal loci in eukaryotic cells.

In accordance with this invention, applicants provide new ways that allow you to apply targeting vectors containing large regions of homology, thus, to modify endogenous genes or chromosomal loci in eukaryotic cells by homologous recombination. Such methods overcome the above limitations of current technologies. In addition, the qualification specialist in this field is clear, the methods of the present invention is readily adaptable for use with any genomic DNA from any eukaryotic organism, including, but not limited to, animals such as mice, rats, other rodents, or humans, and plants, such as soybeans, corn and wheat.

The invention

According to this invention, applicants have developed a new, faster, better and efficient way to create and screenin is and eukaryotic cells which contain modified endogenous genes or chromosomal loci. These new ways to unite for the first time:

1. Bacterial homologous recombination to accurately design the desired genetic modification in a large cloned genomic fragment, obtaining thus a large targeting vector for use in eukaryotic cells (LTVEC).

2. Direct introduction of these LTVEC in eukaryotic cells for the modification of interest endogenous chromosomal locus in these cells.

3. Analysis for determination of rare eukaryotic cells, in which allele-target was modified in the desired manner, providing analysis on the modification of allele (MOA) of the original allele, which does not require information of the sequence outside of the target sequence, such as, for example, quantitative PCR.

The preferred option of the present invention is a method of genetic modification of the endogenous gene or chromosomal locus in eukaryotic cells, providing: (a) obtaining a large cloned genomic fragment containing interest DNA sequence; b) using bacterial homologous recombination to genetically modify this large cloned genomic fragment of (a) to create pain the CSOs targeting vector for use in eukaryotic cells (LTVEC); (C) the introduction of LTVEC (b) in eukaryotic cells for modifying endogenous gene or chromosomal locus in these cells; and (d) using a quantitative assay to detect modification of allele (MOA) in eukaryotic cells (C) to identify those eukaryotic cells in which this endogenous gene or chromosomal locus has been genetically modified.

Another variant of this invention is the way in which genetic modification in relation to the endogenous gene or chromosomal locus includes the deletion of the coding sequence, gene segment, or regulatory element; change the coding sequence, gene segment, or regulatory element; the insertion of new coding sequence, gene segment, or regulatory element; creating a conditional allele; or replacement of the coding sequence or gene segment from one species homologous or ontological coding sequence from another species.

Alternatively, the present invention is the manner in which the change in the coding sequence, gene segment, or regulatory element provides for the replacement, addition or merger where the merger involves epitope label (tag) or a bifunctional protein.

Another option this is about of the invention is a method, where quantitative analysis includes quantitative PCR, comparative genomic hybridization, isothermal amplification of DNA, quantitative hybridization to the immobilized probe, Invader Probes®or MMP-tests®or quantitative PCR incorporates technology with the use of TaqMan probes®, Molecular Beacon or Eclipse™.

Another preferred variant of the present invention is a method in which the eukaryotic cell is an embryonic stem cell of a mammal and, in particular, in which the embryonic stem cell is an embryonic stem cell of a mouse, rat or other rodent.

Another preferred variant of the present invention is a method in which the endogenous gene or chromosomal locus is a gene or chromosomal locus of a mammal, preferably a gene or chromosomal locus of a person or a gene or chromosomal locus of the mouse, rat or other rodent.

Additional preferred variant is a variant in which the LTVEC able to accommodate large DNA fragments by size, greater than 20 TPN, and, in particular, large DNA fragments of a size greater than 100 TPN

Another preferred option is a genetically modified endogenous gene or chromosomal locus, which is produced is by the method of this invention.

Another preferred option is a genetically modified eukaryotic cell, which is obtained by the method of this invention.

The preferred option of the present invention is the body (not people)that contains genetically modified endogenous gene or chromosomal locus obtained by the method of this invention.

Preferred also is the body (not human)derived from genetically modified eukaryotic cells or embryonic stem cells obtained by the method of this invention.

The preferred option is the body (not people)that contains genetically modified endogenous gene or chromosomal locus obtained by the method comprising the stage of: a) obtaining a large cloned genomic fragment containing interest DNA sequence; b) using bacterial homologous recombination to genetically modify a large cloned genomic fragment of (a) to create a large targeting vector (LTVEC) for use in embryonic stem cells; (C) introducing the LTVEC (b) in embryonic stem cells for modifying endogenous gene or chromosomal locus in these cells; (d) use quantitative analysis to detect is odificatio allele (MOA) in embryonic stem cells (C) to identify those embryonic stem cells, in which this endogenous gene or chromosomal locus has been genetically modified; (e) introducing an embryonic stem cell (d) in the blastocyst; and (f) the introduction of a blastocyst (e) a surrogate parent female for gestation.

Additional preferred variant of the present invention is the body (not people)that contains genetically modified endogenous gene or chromosomal locus obtained by the method comprising the stage of: a) obtaining a large cloned genomic fragment containing interest DNA sequence; b) using bacterial homologous recombination to genetically modify a large cloned genomic fragment of (a) to create a large targeting vector for use in eukaryotic cells (LTVEC); (C) introducing the LTVEC (b) in eukaryotic cells for genetic modification of the endogenous gene or chromosomal locus in these cells; and (d) the use of quantitative analysis to detect modification of allele (MOA) in eukaryotic cells (C) to identify those eukaryotic cells in which this endogenous gene or chromosomal locus has been genetically modified; (e) removing the nucleus of eukaryotic cells (d); (f) the introduction of the engine (e) in the oocyte; and (g) the introduction of the oocyte (f) surrogate parents who I female for gestation.

Another preferred option is the body (not people)that contains genetically modified endogenous gene or chromosomal locus obtained by the method comprising the stage of: a) obtaining a large cloned genomic fragment containing interest DNA sequence; b) using bacterial homologous recombination to genetically modify a large cloned genomic fragment of (a) to create a large targeting vector for use in eukaryotic cells (LTVEC); (C) introducing the LTVEC (b) in eukaryotic cells for genetic modification of the endogenous gene or chromosomal locus in these cells; (d) use of quantitative analysis for detect modification of allele (MOA) in eukaryotic cells (C) to identify those eukaryotic cells in which this endogenous gene or chromosomal locus has been genetically modified; (e) merger of eukaryotic cells (d) other eukaryotic cell; and (f) merged introduction eukaryotic cells (e) a surrogate parent female for gestation.

In preferred embodiments, the organism (not man) is a mouse, rat or other rodent; blastocyst blastocyst is a mouse, rat or other rodent; the oocyte is oocyte mouse, rat Il is another rodent; and a surrogate parent is a mouse, rat or other rodent.

Another preferred variant is a variant in which the embryonic stem cell is an embryonic stem cell of a mammal, preferably human embryonic stem cell mouse, rat or other rodent.

Additional preferred option is the use of genetically modified eukaryotic cell of the present invention to obtain the body (not the person) and, in particular, the use of genetically modified embryonic stem cell of the present invention to obtain the body (not the person).

The preferred option of the present invention is a method of genetic modification of interest endogenous gene or chromosomal locus in mouse embryonic stem cells, providing for the stage: a) obtaining a large cloned genomic fragment, more than 20 TPN, which contains interest DNA sequence, where this large cloned DNA fragment is homologous to the endogenous gene or chromosomal locus; b) using bacterial homologous recombination to genetically modify a large cloned genomic fragment of (a) to create a large targeting vector d which I use in mouse embryonic stem cells, where genetic modification is a deletion of the coding posledovatelnosti, gene segment, or regulatory element; (C) the introduction of a large targeting vector (b) in mouse embryonic stem cells for modifying endogenous gene or chromosomal locus in these cells; (d) use quantitative analysis to detect modification of allele (MOA) in mouse embryonic stem cells (C) to identify those embryonic stem cells in which this endogenous gene or chromosomal locus has been genetically modified, where quantitative analysis is quantitative PCR. The preferred option is also genetically modified mouse embryonic stem cell obtained in this way; the mouse that contains a genetically modified endogenous gene or chromosomal locus obtained in this way; and mouse derived from genetically modified mouse embryonic stem cells.

Another preferred option is the mouse that contains genetically modified interest endogenous gene or chromosomal locus obtained by the method comprising the stage of: a) obtaining a large cloned genomic fragment, more than 20 TPN, which contains interest DNA-serial is lnost, where this large cloned DNA fragment is homologous to the endogenous gene or chromosomal locus; b) using bacterial homologous recombination to genetically modify a large cloned genomic fragment of (a) to create a large targeting vector for use in mouse embryonic stem cells, where the genetic modification is a deletion of the coding sequence, gene segment, or regulatory element; (C) the introduction of a large targeting vector (b) in mouse embryonic stem cells for modifying endogenous gene or chromosomal locus in these cells; (d) use quantitative analysis to detect modification of allele (MOA) in mouse embryonic stem cells (C) to identify those mouse stem cells, in which this endogenous gene or chromosomal locus has been genetically modified, where quantitative analysis is quantitative PCR; (e) introduction mouse embryonic stem cell (d) in the blastocyst; and (f) the introduction of a blastocyst (e) as a surrogate of roditelja female for gestation.

Preferred is also the use of genetically modified mouse embryonic stem cells, described above, to obtain the mouse.

Preferred awsumsauce ways in which approximately 1-5 μg large target DNA vector is introduced into approximately 1x107eukaryotic cells.

Brief description of drawings

Figure 1: Schematic diagram of generating a typical LTVEC using bacterial homologous recombination.

(hb1 = block homology 1; hb2 = block homology 2; RE - site restrictase).

Figure 2: Schematic diagram of the donor fragment and LTVEC mouse OCR10.

(hb1 = block homology 1; lacZ = ORF β-galactosidase; SV40 polyA = DNA fragment derived from simian virus 40, contains the site and the polyadenylation signal; PGKp = promoter of the mouse phosphoglycerate (PGK); EM = bacterial promoter; neo = neomycinphosphotransferase; PGK polyA = 3'-untranslated region derived from the PGK gene and contains the site and the polyadenylation signal; hb2 = block homology 2).

Figure 3A-3D: the cDNA Sequence of OCR10 mouse, block homology 1 (hb1), block homology 2 (hb2) and TaqMan probes® and primers used in quantitative PCR analysis to detect modification of allele (MOA) in ES cells using LTVEC mOCR10.

hb1: base pair 1-211

hb2: base pair 1586-1801

The TaqMan probe® and a corresponding set of PCR primers derived from exon 3 mOCR10:

The TaqMan probe®: nucleotides 413-439 - top chain

Primer eh-5': nucleotides 390-410 - top chain

Primer eh-3': the nucleus is the IDA 445-461 - the lower chain

The TaqMan probe® and a corresponding set of primers derived from exon 4 mOCR10:

The TaqMan probe®: nucleotides 608-639 - top chain

Primer EX4-5': nucleotides 586-605 - top chain

Primer EX4-3': nucleotides 642-662 lower chain

Definition

"Targeting vector" is a DNA construct that contains the sequence "homology" endogenous chromosomal sequences of the nucleic acids flanking the desired genetic modification (modification). Flanking sequence homology, called "shoulders homology", direct targeting vector in a specific chromosomal location in the genome due to the homology that exists between the shoulders homology and related endogenous sequence, and enter the desired genetic modification through a process called "homologous modification".

"Homologous" means two or more nucleic acids sequences that are either identical or sufficiently similar to be able to gibridizatsiya to each other or to undergo intermolecular exchange.

"Target gene" is a modification of the endogenous chromosomal locus by insertion into him, deletion from or replacement of the endogenous sequence through homologous is th recombination using a targeting vector.

"Gene knockout is a genetic modification that originate from the destruction of the genetic information encoded in the chromosomal locus.

"Join ("driving gene") is a genetic modification that come from the replacement of genetic information in different chromosomal locus DNA sequence.

"The body with knockout" is a body in which a significant proportion of cells is gene knockout.

"The body is attached ("hammered") the genome is an organism in which a significant proportion of the cells of the body shall be attached ("hammered") gene.

"Marker" or "breeding marker" is a marker for selection, which allows you to select rare transfetsirovannyh cells expressing this marker, most of the treated cells in the population. Genes such markers include, but are not limited to, genes neomycinphosphotransferase and hygromycin In-phosphotransferase or fluorescent proteins such as GFP.

"The ES cell is an embryonic stem cell. This cell is usually derived from the inner cell mass of the embryo at the blastocyst stage.

"A clone of ES cells is a subpopulation of cells derived from single cell populations of ES cells after introduction of the DNA and subsequent selection.

"Flanking DNA" is a DNA segment that is the tsya colinearly and related with a specific point of the reference sequence.

"LTVEC are large targeting vector for eukaryotic cells, which are derived from fragments of the cloned genomic DNA larger than fragments, commonly used in other approaches designed to perform homologous targeting in eukaryotic cells.

"The body (not the man)" is the body, which is usually not perceived publicly as being a person.

"Modification of allele" (MOA) shall mean the modification of the exact DNA sequence of one allele of a gene (genes) or chromosomal locus (loci) in the genome. This modification of allele (MOA) includes, but is not limited to, deletions, substitutions or insertions of just one nucleotide or deletions many TPN that includes the gene of interest (genes) or chromosomal locus (loci), as well as any and all possible modifications between these two extremes.

"Ontologica sequence" means a sequence of one species, which is functionally equivalent to this sequence in a different form.

The description and examples presented infra, are provided to illustrate the present invention. Specialist with expertise in this area will be clear that these examples are provided only as illustrations and are not intended to limit this from the retene.

Detailed description of the invention

Applicants have developed a new, faster, better and efficient way for creating and screening of eukaryotic cells, which contain modified endogenous genes or chromosomal loci. In these cells the modification can be knockouts of the gene (genes), accession (hammering) genes, point mutations or large genomic insertions or deletions, or other modifications. As a non-limiting example, these cells can be embryonic stem cells, which are applicable in the creation of organisms with knockout or joining genes and, in particular, mice with knockout or accession, gene, for the purpose of determining the function of a gene (genes)that have been modified, deleterow and/or insertion.

These new ways to unite for the first time:

1. Bacterial homologous recombination to accurately design the desired genetic modification in a large cloned genomic DNA fragment, obtaining thus a large targeting vector for use in eukaryotic cells (LTVEC);

2. Direct introduction of these LTVEC in eukaryotic cells for the modification of interest of the corresponding endogenous gene (genes) or chromosomal locus (loci) in these cells; and

3. Analysis for determination of rare eukaryotic cells, in which the s allele target was modified in the desired manner, includes quantitative analysis of the modification of allele (MOA) of the original allele.

It should be emphasized that previous methods for detection of successful homologous recombination in eukaryotic cells may be used together with LTVEC of the invention of the authors, as in these LTVEC have large shoulders homology. The use of LTVEC for intentional modification of endogenous genes or chromosomal loci in eukaryotic cells by homologous recombination is possible using a new application of the analysis to determine rare eukaryotic cells, in which allele-target was modified in the desired manner, and this method provides a quantitative analysis of the modification of allele (MOA) of the original allele, using, for example, quantitative PCR or other appropriate quantitative analyses on MOA.

The possibility of using targeting vectors with shoulders homology, greater than the shoulders of homology used in the present methods, is extremely valuable for the following reasons:

1. The target vectors are more quickly and easily generated from the available libraries containing large genomic inserts (e.g. libraries YOU or RACES)than the target vectors obtained the previous ones is the technology, in which genomic inserts must be extensively characterized and "set in order" (cut) before use (which is explained in more detail below). In addition, should be known only minimal information sequence of interest locus, i.e. you only need to know about 80-100 nucleotides that are required to generate blocks of homology (described in detail below) and to generate probes that can be used in quantitative analyses on MOA (described in detail below).

2. Modification of larger size, and modification, including large genomic regions are more easily and with fewer stages generated than with previous technologies. For example, the method of the present invention allows for precise modification of large loci that cannot be absorbed traditional targeting vectors based on the plasmid due to the limitations of their size. It also allows you to modify any particular locus in multiple points (for example, the introduction of specific mutations in different exons multiconsole gene) in one stage, reducing the need to design multiple target vectors and perform multiple rounds of targeting and screening homologous to recombinate is in ES cells.

3. The use of long regions of homology (long shoulder homology) increases the frequency of targeting "hard to target" loci in eukaryotic cells, which is consistent with previous findings that targeting homologous recombination in eukaryotic cells is connected, apparently, with the overall homology contained in the targeting vector.

4. Increased frequency of the target obtained using long shoulder homology, apparently, reduces the usefulness, if any, the use of isogenic DNA in these targeting methods.

5. The application of quantitative analyses MOA for screening of eukaryotic cells by homologous recombination not only allows the use of LTVEC as targeting vectors (advantages outlined above), but also reduces the time required to identify accurately the modified eukaryotic cell, with the usual few days to a few hours. In addition, the use of quantitative MOA does not require the use of probes located outside of the endogenous gene (genes) or chromosomal locus (loci)that are modified, thereby eliminating the necessity of knowledge of the sequences flanking the modified gene (genes) or locus (loci). This represents an important improvement in the way vypolnyaemym in the past, and makes it much less time consuming and much more cost-effective approach to screening event of homologous recombination in eukaryotic cells.

Ways

Many of the methods used to construct DNA vectors described here are standard methods of molecular biology, a well-known specialist with expertise in this area (see, for example, Sambrook, J., E. F. Fritsch And T. Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2, and 3, 1989; Current Protocols in Molecular Biology, Eds. Ausubel et al., Greene Publ. Assoc., Wiley Interscience, NY). All DNA sequencing performed by standard methods using DNA-sequencing machine (ABI 373A and sequencing Taq Dideoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA).

Stage 1. Obtaining a clone large genomic DNA containing the gene of interest (genes) or chromosomal locus (loci).

Interest gene (genes) or locus (loci) can be selected based on specific criteria, such as the detailed structural or functional data, or it can be selected in the absence of such detailed information as potential genes or gene fragments become predictable as a result of efforts of various projects for genome sequencing.

It is important to note that is not necessary the knowledge of the complete sequence and structure of predstavlyayushikh interest of a gene (genes) for the application of the method of the present invention to obtain LTVEC. In fact, the only sequence information is required, are approximately 80-100 nucleotides, so as to gain an interest in the genomic clone, as well as generate homology blocks used in obtaining LTVEC (described in detail below), and to produce probes for use in quantitative analyses MOA.

Upon selection of the gene of interest (gene or locus (loci) receive a large genomic clone (clones)containing this gene (genes) or locus (loci). This clone (clones) can be obtained in any of several ways, including, but not limited to, the appropriate screening of DNA libraries (e.g. libraries YOU, PAC, YAC, or cosmid) standard methods of hybridization or PCR or any other method known to a specialist with expertise in this field.

Stage 2. Join homology blocks 1 and 2 to the modification cassette and generating LTVEC.

Homology blocks mark the sites of bacterial homologous recombination, which are used to generate the LTVEC of large cloned genomic fragments (figure 1). Homology blocks are short segments of DNA, usually double-stranded, and having a length of at least 40 nucleotides that are homologous to regions in the large cloned genomic fragment, fenceroy "area, which mean the inhabitants modification". Homology blocks attached to the modification cassette, so after homologous recombination in bacteria this modification cassette replaces the district, subject to modification (figure 1). The way to create a targeting vector using bacterial homologous recombination can be performed in different systems (Yang et al, Nat Biotechnol, 15:859-65, 1997; Muyrers et al, Nucleic Acids Res, 27:1555-7, 1999; Angrand et al., Nucleic Acids Res, 27:e16, 1999; Narayanan et al., Gene Ther, 6:442-7,1999; Yu, et al., Proc Natl Acad Sci USA, 97:5978-83, 2000). One of the preferred examples of currently used technologies is the NO-cloning (Zhang et al., Nat Genet, 20:123-8, 1998; Narayanan et al., Gene Ther, 6:442-7, 1999) and variations of this technology (Yu, et al., Proc Natl Acad Sci USA, 97:5978-83, 2000). ET denotes proteins recE (Hall and Kolodner, Proc Natl Acad Sci USA, 91:3205-9, 1994) and recT (Kusano et al., Gene,138:17-25,1994), which carry out the reaction of homologous recombination. RecE is an exonuclease that cleaves one chain linear double-stranded DNA (essentially the donor DNA fragment described infra) 5' → 3', leaving behind the linear double-stranded fragment with the 3'single-stranded overhang. This single-stranded protrusion is covered by the recT protein that has the activity of binding single-stranded DNA (ssDNA) (Kovall and Matthews, Science, 277:1824-7, 1997). ET-cloning is carried out using E. coli, which temporarily expresses gene products recE and recT E. coli (Hall and Kolodner, Froc Natl Acad Sci USA, 91:305-9, 1994; Clark et al., Cold Spring Harb Symp Quant Biol, 49:453-62, 1984; Noirot and Kolodner, J Biol Chem, 273:12274-80, 1998; Thresher et al., J Mol Biol, 254:364-71, 1995; Kolodner et al., Environ Mol, 11:23-30, 1994; Hall et al., J Bacteriol, 175:277-87, 1993), and protein λgam bacteriophage lambda (λ) (Murphy, J Bacteriol,173:5808-21, 1991; Poteete et al., J Bacteriol, 170:2012-21, 1988). Protein λgam necessary to protect the donor DNA fragment from degradation system ectonucleoside recBC (Myers and Stahl, Annu Rev Genet, 28:49-70, 1994), and it is necessary for the effective ET-cloning in recBC+hosts, such as the commonly used E. coli strain DH10b.

The area is subject to modification and replacement using bacterial homologous recombination, can be in the range from zero nucleotides in length (creating an insertion in the original locus) to many tens of TPN (creating deletion and/or replacement of the initial locus). Depending on the modification tapes, this modification may result in the following:

(a) deletion of the coding sequence, gene segment, or regulatory element;

(b) change (change) the coding sequence, gene segment, or regulatory elements, including substitutions, additions and mergers (e.g., epitope tags, or the creation of bifunctional proteins, such as proteins with CFP);

(C) inserting a new coding regions, gene segment, or regulatory elements, such as coding regions, gene is egment or regulatory elements for breeding marker genes or reporter genes, or putting new genes under the transcriptional control of the endogenous;

(d) creation of conditional alleles, for example, the introduction of loxP sites flanking the region to be cut Cre-recombinase (Abremski and Hoess, J Biol Chem, 259:1509-14, 1984), or FRT sites flanking the region to be cut Flp-recombinase (Andrews et al., Cell 40:795-803, 1985; Meyer-Leon et al., Cold. Spring Harb Symp Quant Biol, 49:797-804, 1984; Cox, Proc Natl Acad Sci USA, 80:4223-7, 1983); (e) replacing the coding sequence or gene segments from one species ontologyname coding sequences from different species, for example, replacement of mouse genetic locus ontological genetic locus person to construct a mouse, in which this particular locus is "humanized".

Any of these modifications can be included in the LTVEC. Specific, non-limiting example, in which the coding sequence of the endogenous fully demeterova and simultaneously replaced as reporter gene, and breeding marker, is provided below in example 1, as well as the advantages of the method of the present invention in comparison with the previous technologies.

Stage 3 (optional). Confirmation that each LTVEC was designed correctly.

Confirmation that each LTVEC was designed correctly, get through:

A. Diagnostic PCR for p is dorigine new connections, created by the introduction of donor fragment in the gene of interest (genes) or chromosomal locus (loci). Thus obtained PCR fragments can be sequenced for further confirmation of new connections created by the introduction of donor fragment in the gene of interest (genes) or chromosomal locus (loci).

b. Diagnostic splitting restrictable to obtain reasonable assurance that only the desired modifications have been introduced in the LTVEC during bacterial homologous recombination.

C. Direct sequencing LTVEC in particular areas, including site modifications, to confirm your new connections created by the introduction of donor fragment in the gene of interest (genes) or chromosomal locus (loci).

Stage 4. Cleaning, receiving and linearization LTVEC DNA for introduction into eukaryotic cells.

A. Obtaining DNA LTVEC:

Get minireport DNA (Sambrook, J., E. F. Fritsch And T. Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2, and 3, 1989; Tillett and Neilan, Biotechniques, 24:568-70, 572, 1998; http://www.qiagen.com/ literature/handbooks/plkmmi/plm_399.pdf) selected LTVEC and re-transform minireport LTVEC DNA in E. coli using electroporation (Sambrook, J., E. F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2, and 3, 1989). This stage is necessary to remove the plasmid coding recombinogenic proteins, the cat is who are used to stage the bacterial homologous recombination (Zhang et al., Nat Genet, 20:123-8, 1998; Narayanan et al., Gene Ther, 6:442-7, 1999). From this plasmid is useful to get rid of (a), as it is vysokonapornoj a plasmid and can reduce the outputs obtained in large-scale preparations LTVEC; (b) to exclude the possibility of induction of expression of recombinogenic proteins; and (C) because it can hinder the physical mapping of LTVEC. Before introducing the LTVEC in eukaryotic cells receive large numbers of LTVEC DNA standard methodology (http://www.qiagen.com/literature/handbooks/plk/ plklow.pdf; Sambrook, J., E. F. Fritsch And T. Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2, and 3, 1989; Tillett and Neilan, Biotechniques, 24:568-70, 572, 1998). However, this stage can be neglected when using the method of the bacterial homologous recombination using recombinogenic of propaga, i.e. when the genes encoding recombinogenic proteins integrated into the bacterial chromosome (Yu, et al., Proc Natl Acad Sci USA, 97:5978-83, 2000).

b. Linearization LTVEC DNA:

To obtain LTVEC for introduction in eukaryotic cells LTVEC preferably linearized in a way that leaves DNA modified endogenous gene (genes) or chromosomal locus (loci), flanked long shoulders homology. This can be done by linearization LTVEC, preferably in vector frame, any restricted, which decompose only rarely. Examples of suitable restricts include NotI, PacI, SfI, SrfI, SwaI, FseI, etc. the Choice of restriction enzyme can be determined experimentally (i.e. test several different candidates as rarely splitting of restrictus) or if the sequence of this LTVEC known, sequence analysis and selection of the appropriate restriction enzymes on the basis of this analysis. In situations where LTVEC has vector frame containing the rare sites of cleavage, such as sites CosN, it can be broken down by enzymes that recognize these sites, for example, λ-terminate (Shizuya et al., Proc Natl Acad Sci USA, 89:8794-7, 1992; Becker and Gold, Proc Natl Acad Sci USA, 75:4199-203, 1978; Rackwitz et al., Gene, 40:259-66, 1985).

Stage 5. Introduction LTVEC in eukaryotic cells and selecting cells in which was successful introduction of the LTVEC.

LTVEC DNA can be introduced into eukaryotic cells using standard methodology, such as transfection mediated by calcium phosphate, lipids, or electroporation (Sambrook, J., E. F. Fritsch And T. Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1,2, and 3, 1989). Cells, in which LTVEC was introduced successfully, can be selected by exposure to the action of the agents of selection, depending on breeding marker gene, which was built in LTVEC. As a non-limiting example, if breeding marker is neomycinphosphotransferase gene (neo) (Beck, et al., Gene, 19:327-36, 1982), the cells that are included in LTVEC, can be selected on G418-containing medium, the cells that are not included LTVEC, will die, whereas cells that included LTVEC will survive (Santerre, et al., Gene, 30:147-56, 1984). Other suitable breeding markers include every drug that has activity in eukaryotic cells (Joyner, The Practical Approach Series, 293, 1999), as well as hygromycin In (Santerre, et al., Gene, 30:147-56, 1984; Bernard, et al., Exp Cell Res, 158:237-43, 1985; Giordano and McAllister, Gene, 88:285-8, 1990), Blasticidin S (Izumi, et al., Exp Cell Res, 197:229-33, 1991) and others that are well-known specialists with expertise in this field.

Stage 6. The screening event of homologous recombination in eukaryotic cells using quantitative analysis, a modification of allele (MOA).

Eukaryotic cells that have been successfully modified by targeting LTVEC in interest locus can be identified using a variety of approaches that can be used to detect modification of allele in interest locus and which do not depend on analyses covering the full shoulder homology or full shoulders homology. Such approaches may include, but are not limited to:

(a) quantitative PCR using TaqMan® (Lie and Petropoulos, Curr Opin Biotechnol, 9:43-8, 1998);

(b) quantitative analysis of the MOA with the use of molecular beacons (Tan, et al, Chmistry, 6:1107-11, 2000);

(C) hybridization with fluorescence in situ FISH (Laan, et al, Hum Genet, 96:275-80, 1995) or comparative genomic hybridization (CGH) (Forozan et al, Trends Genet, 13:405-9, 1997; Thompson and Gray, J Cell Biochem Suppl, 139-43, 1993; Houldsworth and Chaganti, Am J Pathol, 145:1253-60, 1994);

(d) the isothermal DNA amplification (Lizardi, et al, Nat Genet, 19:225-32, 1998; Mitra and Church, Nucleic Acids Res, 27:e34, 1999);

(e) quantitative hybridization to the immobilized probe (probes) (Southern, J. Mol. Biol. 98: 503, 1975; Kafatos FC; Jones CW; Efstratiadis A, Nucleic Acids Res 7(6):1541-52, 1979);

(f) Invader Probes® (probes invaders) (Third Wave Technologies); (Technologies of the Third Wave);

(g) Eclipse™ and Molecular probes-beacons (Synthetic Genetics); and

(h) MMP-analyses (High Throughput Genomics).

The authors of the invention provide here an example in which quantitative PCR with TaqMan® used for screening successfully targeted eukaryotic cells. In this non-limiting example, TaqMan® used for identification of eucaryotic cells which have undergone homologous recombination, in which a part of one of the two endogenous alleles in a diploid genome has been replaced by another sequence. In contrast to traditional methods, in which the difference in the length of restriction fragments, including all the shoulder (or shoulders) homology indicates a modification of one of the two alleles of a quantitative method using TaqMan® will detectorbut is a modification of one allele by measuring the decrease in the copy number of (half) unmodified allele. Specifically, this probe detects unmodified allele and not the modified allele. Thus, this method does not depend on the exact nature of the modification and is not limited to the replacement of the sequence described in this example. TaqMan® used to quantify the number of copies of DNA template in a sample of genomic DNA, in particular, by comparison with a reference genome (Lie and Petropoulos, Curr Opin Biotechnol, 9:43-8, 1998). The reference gene is quantitatively determined in the same genomic DNA in which the gene target (genes) or locus-target (loci). Therefore, performs two TaqMan®amplification (each with its corresponding probe). One probe TaqMan® defines the Ct (threshold cycle) of the reference gene, while the other probe determines the Ct region of the target genes (genes) or locus-target (loci), which replaced the successful targeting. Ct is the amount that reflects the amount of starting DNA for each TaqMan probes®i.e. less presents the sequence requires more PCR cycles to reach the threshold cycle. Reducing by half the number of copies of the matrix sequence for the reaction TaqMan® will lead to an increase of approximately one Ct unit. The reaction TaqMan® in cells in which one allele of the target genes (genes) or locus-target (loci) was replaced with the homologous re is combinatii, will lead to increase of one Ct unit for TaqMan®reaction of the target genes without increasing Ct for the reference gene when compared with DNA from cells without the successful targeting. This makes it easy to detect the modification of one allele gene of interest (genes) in eukaryotic cells using LTVEC.

As noted above, screening modification of allele (MOA) is the use of any method, which detects a modification of one of the alleles for identification of cells that have undergone homologous recombination. It does not require alleles target were identical (homologous) to each other, and, in fact, they may contain polymorphisms, as is the case in the progeny derived from crossing two different strains of mice. In addition, one special situation, which is also covered by the screening MOA is targeting genes that normally are present in a single copy in the cell, such as some localized on the sex chromosome genes, and, in particular, on the Y-chromosome. In this case, the methods that will detect the modification that only one allele of the target, such as quantitative PCR, blotting on the Southern, etc. can be used to detect events targeting. It is clear that the method of this invention can be used for the van to generate the modified eukaryotic cell even in the case when alleles are polymorphic or when they are present as a single copy in the target cells.

Stage 8. The use of genetically modified eukaryotic cell.

(a) Genetically modified eukaryotic cells generated by the methods described in stages 1-7, can be used in any analysis in vitro or in vivo, where it is desirable modification of the phenotype of such cells.

(b) Genetically modified eukaryotic cells generated by the methods described in stages 1-7, can also be used to generate an organism carrying a genetic modification. Genetically modified organisms can be obtained in several different ways, including, but not limited to: 1. Modified embryonic stem (ES) cells, such as those commonly used ES cells from rats and mice. ES cells can be used to create genetically modified rats or mice standard technology with blastocyst injection or aggregation (Robertson, Practical Approach Series, 254, 1987; Wood, et al., Nature, 365:87-9, 1993; Joyner, The Practical Approach Series, 293, 1999), injection tetraploid blastocysts (Wang, et al., Mech Dev, 62:137-45,1997),or by transfer of nuclei and cloning (Wakayama, et al., Proc Natl Acad Sci USA, 96:14984-9, 1999). ES cells derived from other organisms, such as rabbits (Wang, et al., Mech Dev, 62137-45, 1997; Schoonjans, et al., Mol Reprod Dev 45:439-43, 1996) or chickens (Pain et al., Development, 122:2339-48, 1996) or other species should also be suitable for genetic modification (modification) using the methods of the present invention.

2. Modified protoplasts can be used to generate genetically modified plants (for example, see U.S. patent 5350689 "Zea mays plants and transgenic Zea mays plants regenerated from protoplasts or protoplast-derived cells", and U.S. patent 5508189 "Regeneration of plants from cultured guard cell protoplasts and references in them).

3. The transfer of nuclei from the modified eukaryotic cells into oocytes to generate cloned organisms with modified allele (Wakayama, et al., Proc Natl Acad Sci USA, 96:14984-9, 1999; Baguisi et al., Nat Biotechnol, 17:456-61, 1999; Wilmut, et al., Reprod Fertil Dev 10:639-43, 1998; Wilmut, et al., Nature, 385:810-3, 1997; Wakayama, et al., Nat Genet, 24:108-9, 2000; Wakayama et al., Nature, 394:369-74, 1998; Rideout, et al., Nat Genet, 24:109-10, 2000; Campbell, et al., Nature, 380:64-6, 1996).

4. Merge cells to migrate the modified allele in the other cell, which includes the transfer of engineered chromosomes (chromosomes), and the use of such cells (cells) to generate organisms bearing the modified allele or constructed chromosome (chromosome) (Kuroiwa, et al., Nat Biotechnol, 18:1086-1090, 2000).

5. The method of the present invention is also applicable to any other approaches that have been used or will be opened.

Although many of the methods that are used the most in the practice of individual stages of the methods of the present invention, are known for a specialist with expertise in this area, the applicants argue that the novelty of the method of the present invention lies in the unique combination of these stages and methods associated with never-before-described method of introducing the LTVEC directly into eukaryotic cells for modification of a chromosomal locus, and the use of quantitative MOA assays for the identification of eukaryotic cells, which have been appropriately modified. This new combination represents a significant improvement over previous technologies to create organisms with modifications of endogenous genes or chromosomal loci.

Examples

Example 1: Construction of mouse ES cells carrying a deletion of the gene OCR10.

A. The selection of clone large genomic DNA containing mOCR10.

Clone bacterial artificial chromosome (BAC)carrying large fragment of genomic DNA that contains the coding sequence of the murine gene OCR10 (mOCR10), was obtained by screening matrix library YOU mouse genomic DNA (Incyte Genomics) using PCR. The primers used for the screening of this library were produced from the cDNA sequence of the gene mOCR10.

Used two pairs of primers:

(a) OCR10.RAA (5'-AGCTACCAGCTGCAGATGCGGGCAG-3') and

OCR10.PVIrc (5'-CTCCCCAGCCTGGGTCTGAAAGATGACG-3'), which amplificare D Is the size of 102 BP; and

(b) OCR10.TDY (5'-GACCTCACTTGCTACACTGACTAC-3') and OCR10.QETrc (5'-ACTTGTGTAGGCTGCAGAAGGTCTCTTG-3', which amplificare DNA size 1500 BP

This mOCR10 YOU contained approximately 180 TPN genomic DNA, including the complete coding sequence mOCR10. This YOU clone used to generate the LTVEC, which is then used for deletion of part of the coding region mOCR10 while also introducing a reporter gene, initiating codon which just replaced the initiating codon OCR10 and insertion breeding gene marker used for selection in E. coli and in mammalian cells, after the reporter gene (figure 2). This reporter gene (in this non-restrictive example, LacZ, the sequence of which is easily accessible to the person skilled in the art) that encodes the enzyme β-galactosidase of E. coli. Due to the provisions of the LacZ insertions (his initiating codon is in the same position as the initiating codon mOCR10) expression of lacZ should mimic the expression of mOCR10, as observed in other examples, where he performed such replacement using LacZ using older technologies (see "Gene trap strategies in ES cells", by W Wurst and A. Gossler, in Joyner, The Practical Approach Series, 293, 1999). Gene LacZ, you can perform simple and standard enzymatic analysis, which can reveal a picture of his expression in situ, thus providing a surrogate for the initial analysis, which reflects the picture of the normal expression replaced by a gene (genes) or chromosomal locus (loci).

b. Construction of the donor fragment and generating LTVEC.

Modification cassette used in the construction of LTVEC mOCR10 is cassette lacZ-SV40 polyA-PGKp-EM7-neo-PGK polyA, where lacZ is a marker gene described above, SV40 polyA is the fragment produced from simian virus 40 (Subramanian, et al., Prog Nucleic Acid Res Mol Biol, 19:157-64, 1976; Thimmappaya et al., J Biol Chem, 253:1613-8, 1978; Dhar, et al., Proc Natl Acad Sci USA 71:371-5, 1974; Reddy, et al., Science, 200:494-502, 1978) and contains the site and the polyadenylation signal (Subramanian, et al., Prog Nucleic Acid Res Mol Biol, 19:157-64, 1976; Thimmappaya et al., J Biol Chem, 253:1613-8, 1978; Dhar, et al., Proc Natl Acad Sci USA 71:371-5, 1974; Reddy, et al., Science, 200:494-502, 1978), PGKp is the promoter of the mouse phosphoglycerate (PGK) (Adra et al., Gene, 60:65-74, 1987) (which is used widely to initiate the gene expression of drug resistance in mammalian cells), EM7 is a strong bacterial promoter, which has the advantage that it allows positive selection in bacteria full design LTVEC by running the neomycinphosphotransferase gene (neo)neo is a breeding marker, which confers resistance to kanamycin in prokaryotic cells and resistance to G418 in eukaryotic cells (Beck, et al., Gene, 19:327-36, 1982), and olyA PGK is a 3'-untranslated region, made from PGK gene and contains the site and the polyadenylation signal (Boer, et al., Biochem Genet, 28:299-308, 1990).

To construct LTVEC mOCR10 first generated donor fragment, consisting of a block of homology 1 (hb1) mOCR10 attached to the left from the LacA gene modification in the cassette, and a block of homology 2 (hb2) mOCR10 attached to the right of the polyA sequence neo-PGK in the modification cassette (figure 2), using standard techniques of recombinant genetic engineering. Block homology 1 (hb1) consists of 211 BP noncoding sequence directly to the left of the initiating methionine of the open reading frames mOCR10 (mOCR10 ORF) (figure 3A-3D). Block homology 2 (hb2) consists of the last 216 BP ORF mOCR10 ending with the stop codon (figure 3A-3D).

Then, using bacterial homologous recombination (Zhang, et al., Nat Genet, 20:123-8, 1998; Angrand, et al., Nucleic Acids Res, 27:e16, 1999; Muyrers et al., Nucleic Acids Res, 27:1555-7, 1999; Narayanan, et al., Gene Ther, 6:442-7, 1999; Yu, et al., Proc Natl Acad Sci USA, 97:5978-83, 2000), this donor fragment used for exact replacement of the coding region mOCR10 (from the initiator methionine to stop codon) insertional cassette, which led to the design of LTVEC mOCR10 (figure 2). Thus, in this LTVEC mOCR10 the coding sequence mOCR10 was replaced by the insertion of the cassette from the creation to the deletion of approximately 20 TPN in the locus mOCR10 with what is given at the same time, approximately 130 TPN homology to the left (left shoulder homology) and 32 TPN homology to the right (right shoulder homology).

It is important to note that LTVEC can be more quickly and easily generated from the available libraries YOU than targeting vectors obtained using the previous technology, as it requires only one stage of bacterial homologous recombination and the only required sequence information is information sequence necessary to generate blocks of homology. In contrast, previous approaches to generate targeting vectors using bacterial homologous recombination require large target vectors were "pruned" before their introduction into ES cells (Hill et al., Genomics, 64:111-3, 2000). This trimming is necessary due to the need of generating shoulders homology, short enough to adapt to the ways of screening used by previous approaches. One of the main disadvantages of the method of Hill et al. is that it requires two additional stages of homologous recombination just for cropping (one for trimming the area to the left of the modified locus and one for trimming the area to the right of the modified locus). To do this, it requires considerably more information about the sequence, including Chi the Le sequence information, includes sites cropping.

In addition, another obvious advantage, as illustrated by the above example, is that a very large deletion that includes the gene mOCR10 (approximately 20 TPN), can be easily generated with a single stage. In contrast, the old technology may require to perform the same task several stages and may include marking the areas to the left and to the right of the coding sequences of loxP sites for the application of Cre-recombinase to remove sequences that are flanked by these sites, after the introduction of the modified loci in eukaryotic cells. This may be impossible in a single stage, and, therefore, may require the construction of two targeting vectors using different selection markers, and two successive events targeting in ES cells, one for introduction of site lozP in the area to the left of the coding sequence and the other for the introduction of the loxP site in the area to the right of the coding sequence. Further it should be noted that the creation of large deletions often occurs with low efficiency when using previous technologies targeting in eukaryotic cells, as the frequency of receiving homologous recombination can be low when using n tselevaya vectors, containing a large deletion, flanked relatively short shoulders homology. High efficiency obtained using the method of the present invention (see below), due to very long shoulders homology present in the LTVEC, which increase the rate of homologous recombination in eukaryotic cells.

C. Confirmation, the receipt and entry of the LTVEC DNA mOCR10 in ES cells.

The sequence surrounding the junction of the insertion of the cassette, and the sequence homology was confirmed by DNA sequencing. The size of the LTVEC mOCR10 was confirmed by restriction analysis and subsequent electrophoresis pulsed electric field (PFGE) (Cantor, et al., Annu Rev Biophys Biophys Chem 17:287-304, 1988; Schwartz and Cantor, Cell 37:67-75, 1984). Performed standard large-scale obtaining plasmids LTVEC mOCR10, plasmid DNA was digested with restriction enzyme NotI, which produces an incision in the carcass of the vector LTVEC mOCR10 with the formation of linear DNA. Then the linearized DNA was introduced into mouse ES cells by electroporation (Robertson, Practical Approach Series, 254, 1987; Joyner, The Practical Approach Series, 293, 1999; Sambrook, et al., Sambrook, J., E. F. Fritsch and T. Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2, and 3, 1989). ES cells, successfully transfetsirovannyh LTVEC mOCR10, were selected in G418-containing medium using standard selection methods (Robertson, Practical Approach Series, 254, 1987; Joyner, The Practical Approach Series, 293, 1999).

d. Identifying acacia ES cells with successful targeting using quantitative analysis modification of allele (MOA).

To identify ES cells in which one of the two endogenous genes mOCR10 was replaced by sequence modification cassettes, DNA from individual clones of ES cells were analyzed by quantitative PCR using standard TaqMan®-methodology as described (Applied Biosystems, TaqMan® Universal PCR Master Mix, catalog number P/N 4304437; see also http://www.pebiodocs.com/pebiodocs/04304449.pdf). The used primers and TaqMan®-probes are the same as described for figure 3A-3D. Were skanirovaniya overall, 69 independent clones of ES cells and 3 of them were identified as positive, i.e. as clones, in which one of the endogenous coding mOCR10 sequence was replaced with the above-described modification cassette.

Several advantages of the MoA approach are obvious:

(i) It does not require the use of a probe out to be modified locus, which eliminates the need for knowledge of the sequences flanking the modified locus.

(ii) It requires little time to complete compared with the conventional methodology blotting for Southern, which was previously the preferred method (Robertson, Practical Approach Series, 254, 1987, Joyner, The Practical Approach Series, 293, 1999), reducing, therefore, the time to identify correctly modified cells with normal a few days to a few hours.

This is a significant superior who eat the way screening, performed in the past, and makes this method less time consuming and more cost-effective approach to screening event of homologous recombination in eukaryotic cells.

Another advantage of the method of the present invention is that it also exceeds the previous technology due to its ability to make targeting difficult to target loci. Using previous technologies, it was shown that for certain loci, the frequency of successful targeting can be as low as 1 in 2000 event integration, perhaps even lower. Using the method of the present invention, the applicants have demonstrated that such a difficult loci can discover much more effective targeting using LTVEC that contain long shoulders homology (i.e. longer than shoulders homology, possible in the case of the former technologies). As a non-limiting example, the above demonstrates that the applicants had complied with the targeting locus OCR10, locus, which, as was previously shown, is not amenable to targeting by using the conventional technology. Using the method of the present invention, applicants have shown that they have been successful targeting in 3 of 69 clones of ES cells, which was integrated LTVEC mOCR10 (containing breeches 160 TPN shoulders homology and introducing a deletion of 20 TPN), whereas using the old technology for targeting ES cells (Joyner, The Practical Approach Series, 293, 1999) using vector-based plasmid with a shrug homology, shorter than 10-20 TPN, deletions of less than 15 TPN though was administered, were not identified event targeting among more than 600 integrants of this vector. These data clearly demonstrate the superiority of the method of the present invention over previous technologies.

Example 2: Increased frequency targeting and eliminating the necessity of the use of isogenic DNA using LTVEC as targeting vectors.

As noted above, the increased frequency of the target obtained using long shoulder homology, should reduce the benefit, if any, obtained from the application of the genomic DNA in the construction of LTVEC, which is isogenic (i.e. identical in sequence) with the DNA of eukaryotic target cells. To test this hypothesis, applicants have designed several LTVEC using genomic DNA obtained from the same subschema mouse, derived from the eukaryotic cell to be targeting (presumably isogenic), and a large number of other LTVEC using genomic DNA obtained from mouse substantiv different from subschema eukaryotic CL the weave be targeting (presumably nesoenas). Netgenie LTVEC showed the average rate target of 6% (in the range of 1-20%, table 1), whereas the isogenic LTVEC showed the average rate targeting 3% (in the range 2-5%), which suggests that the frequency of successful targeting using LTVEC does not depend on usagenote.

Example 3: a Detailed description of the analysis of the MOA based on TaqMan® to identify the ES clones with successful targeting.

Clones of ES cells that have absorbed the LTVEC and turned it into the genome at the locus targeted homologous recombination, identify analysis modification of allele (MOA), which uses quantitative real-time PCR to detect differences between clones of ES cells with successful targeting, in which one of the two alleles of the target is modified, and clones of ES cells without the successful targeting, in which both alleles remain unaltered. Analysis of the MOA consists of primary and secondary screening. Primary screening includes the following stages: (1) cultivation of LTVEC-transfected clones of ES cells on coated gelatin 96-well tablets; (2) isolation of genomic DNA from each clone ES-cells; (3) using each sample of genomic DNA as template in 8 separate quantitative PCR in two 384-well p is anseth, which 2 of these PCR using a set of specific locus-target primers that hybridize to DNA sequences on one side genomic fragment aimed at a deletion ("left-PCR"), 2 of these PCR using a set of specific locus-target primers that hybridize to DNA sequences on the other side genomic fragment aimed at a deletion ("right-PCR"), 4 of PCR using a set of primers that recognize four no target reference locus ("reference PCR"), and each PCR includes fluorescent probe (e.g., TaqMan® [ABI], Eclipse™ or Molecular makovy probe [Synthetic Genetics]), that recognizes the amplified sequence and the fluorescence signal which is directly proportional to the amount of PCR product; (4) the holding of these PCR device that is a combination of thermal cycler and fluorescence detector (for example, ABI 7900HT), which determines the accumulation of amplification products during PCR and determines the threshold cycle (CT), the point in PCR, in which the fluorescence signal is detektivami above background noise; (5) for the sample DNA of each clone ES-cells calculating the differences in the values of CT(ΔCTbetween "left" PCR and each of the four reference PCR and between right PCR and each of the four reference PCR to create the of 8 tables of 96 units Δ CT; (6) the normalization values ΔCTrelatively positive values; (7) the calculation of the median value ΔCTfor each table comparing the target-reference locus; (8) determination of the confidence level with the help of a computer program, which has eight tables ΔCTand calculates, in which the number of cases in the sample DNA clone ES-cells gives a higher value ΔCTin tolerance (permissible) within 0,5-1,5, 0,25-1,5, 0,5-2,0, 0,25-2,0, 0,5-3,0 and 0.25 to 3.0 cycles than the median ΔCT(examples of languages of computer programming, suitable for making or recording of such programs include visual basics, Java or any other computer programming language, well-known specialist with expertise in this area); (9) the construction of these units and their medians for each of the eight tables ΔCTin the form of histograms; and (10) identification of clones candidate ES cells with the correct targeting of the consideration of the confidence levels and histograms ΔCT. In a preferred example, the value of ΔCTfor clones candidates with successful targeting is in the range of 0.5 to 1.5 cycles greater than the median in 8 of 8 reference comparisons.

Clones candidates identified by the initial screening analysis of MOA, may accept or reject in the secondary screening, to the which includes the following stages: (1) using genomic DNA from each of the positive clones candidate ES cells, from a larger number of negative clones and standard number of copies of genomic DNA from mice that carry one or two copies of the cassette LacZ-Neo LTVEC for diploid, as matrices in 8 separate quantitative PCR in two 384-well tablets, 1 in which the reaction is "left" PCR (as in the primary screening), one reaction is "right" PCR (as in the primary screening), 4 reactions are reference PCR with two reference loci that differ from the reference loci used in the primary screening, one PCR reaction is with the primers and probes, which are specific for the LacZ gene LTVEC, and one reaction is a PCR with primers and probes, which are specific for the Neo gene LTVEC; (2) the holding of these PCR device for quantitative PCR, as in the primary screening; (3) calculation, as in the primary screening values (ΔCTbetween "left" PCR and each of the two reference PCR, between "right" PCR and each of the two reference PCR, between LacZ PCR and each of the reference between PCR and PCR Neo and each of the two reference PCR to create eight tables ΔCT; (4) normalization of the values ΔCTrelatively positive values; (5) the calculation of medians for each table ΔCT; (6) calculation of confidence levels, as in the primary screening; (7) the construction of these variables and their median d the I each of the eight tables Δ CTin the form of histograms.

From consideration of the confidence levels and histograms ΔCTas for primary and secondary screening clones candidate ES cells with the correct targeting either confirmed or rejected. In a preferred example, the value of ΔCTfor clones candidates with successful targeting is in the range of 0.5 to 1.5 cycles greater than the median in 12 of 12 reference comparisons of the combined primary and secondary screening.

To estimate the number of copies LTVEC for diploid confirmed in ES clones with the correct target values ΔCTfrom comparisons of PCR LacZ and Neo with two reference PCR compared with values ΔCTfor standards number of copies of the LacZ-Neo. Each clone ES-cells assessed as having 1, 2 or more than 2 copies of LTVEC. For each modified allelic product clones of ES cells subjected to screening in groups of 96 (usually less than 288 clones in total)until you have identified 3 clone, which are assessed as positive in the analysis of the MoA and have the only copy of the tape LacZ-Neo.

Example 4: Application of FISH hybridization with fluorescence in situ) to identify correctly targeted LTVEC in ES cells.

Using technology LTVEC described herein, applicants have destroyed gene SM22 in ES cells. SM22 is Belk is m Ng 22 kDa, limited line of differentiation of smooth muscle cells (SMC), which physically connects the bundles of filaments of actin cytoskeleton in contractile SMC. Then ES cells with successful targeting subjected to standard hybridization with fluorescence in situ (FISH) to metaphase chromosomal distributions to confirm that the target gene was correct. This experiment was performed with two probes: 1) probe gene SM22 consisting of unmodified clone YOU SM22 used to generate the LTVEC, and 2) the probe DNA LacZ and neomycin, which detects only the modification of a gene produced by the event target (interturbine cassettes of genes LacZ and Neo). Metaphase chromosome distribution was obtained from cells, and hybridization was performed simultaneously with both probes, which were labeled with a different colored fluorophores to allow detection of hybridization of each probe in the same chromosomal distribution. Line ES cells without targeting was analyzed in parallel as a control. As expected, in the control of chromosomal distributions of two alleles SM22 were detected on the homologous chromosome shoulders, but no hybridization probe LacZ-Neo. As controls, the distributions of ES cells with the targeting two alleles were detected only in the same chromosome the m position on homologous chromosomes but double tagging probe LacZ-Neo was visible on one of the two chromosomes, indicating that the co-localization of DNA sequences SM22 and LacZ-Neo in this allele SM22. It is important that the DNA sequence of a gene SM22 or genes LacZ-Neo is not detected in the wrong locations in these chromosomal distributions. No extraenteric sequences of the gene SM22 and co-localization of LacZ-Neo with SM22 in one chromosome of the homologous pair largely assumes that took place the correct targeting LacZ-Neo on one of the alleles SM22 through homologous recombination.

Example 5: reducing the amount of DNA used for electroporation of ES cells, improves the efficiency target.

Standard methods for targeted gene modifications in mouse embryonic stem (ES) cells typically use 20-40 µg target vector in the electroporation procedure. The inventors have found that using LTVEC electroporation with much lower amounts of DNA in the range of from about 1 to 5 μg per 1x107cell - doubling of frequency correctly targeted homologous recombination events, greatly reducing the number of secondary, non-homologous insertion events. This is a clear improvement in the efficiency of targeting is important, that is how it significantly reduces the number of clones of ES cells, to be subject to screening to find several positive clones with correctly targeted odnostadiinoi modification. Related benefits are reduced cost and increased performance of the method.

Example 6: the Application of the method of the invention to create mice with knockout MA to study muscle atrophy.

MA, also called MAFbx, is a newly open ubiquitinate, which is negatively regulated by the various States of muscle atrophy (See. Provisional application U.S. No. 60/264926, filed January 30, 2001, Provisional application U.S. No. 60/311697, filed August 10, 2001, and Provisional application U.S. (registration number not yet known), filed on October 22, 2001, all assigned Regeneron Pharmaceuticals, Inc., each of which is incorporated herein in its entirety by reference). To further study the biological significance of this gene in muscle atrophy created mice with knockout of the gene using the method of the present invention as follows.

First, for obtaining a large cloned genomic fragment containing the gene MA, library of bacterial artificial chromosomes (BAC) was subjected to screening with primers produced from cDNA sequences MA. Thus obtained clone YOU IP is was olovely then to create a Large Targeting Vector for Eukaryotic cells (LTVEC) as follows. Designed modification cassette containing the 5'-block homology/gene lacZ/polyA/promoter PGK/neo/polyA/3'-block homology. Homology blocks were added to mark the sites of bacterial homologous recombination during the generation LTVEC. LacZ is a reporter gene, which was placed so that its initiator codon was in the same position as the initiating codon ME. After homologous recombination in bacteria this modification cassette was replaced with the gene MA. Thus was created the LTVEC MA, where the coding sequences MA in clone YOU replaced the modification cassette constructed as described above. Then got LTVEC DNA was purified and was linearizable for introduction in eukaryotic cells, as described infra.

Received minireport LTVEC DNA MA (Sambrook, J., E. F. Fritsch And T. Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2, and 3, 1989; Tillett and Neilan, Biotechniques, 24:568-70, 572, 1998; http://www.qiagen.com/ literature/handbooks/plkmini/plm_399.pdf) and re-transformed into E. coli using electroporation (Sambrook, J., E. F. Fritsch and T. Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2, and 3, 1989) to remove the plasmid coding recombinogenic proteins that are used for stage bacterial homologous recombination (Zhang et al., Nat Genet, 20:123-8, 1998; Narayanan et al., Gene Ther, 6:442-7, 1999). Before introducing the LTVEC MA in eukaryotic cells large number of LTVEC MA received from andartes methodology (http:,,www.qiagen.com/literature/ handbooks/plk/plklow.pdf; Sambrook, J, E. F. Fritsch And T. Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2 and 3, 1989; Tillett and Neilan, Biotechniques, 24:568-70, 572, 1998).

Then, to obtain the LTVEC MA for introduction in eukaryotic cells LTVEC ME was linearizable. This was done by splitting the restriction enzyme NotI, resulting in DNA modified endogenous gene (genes) or chromosomal locus (loci), flanked long shoulders homology.

Then LTVEC MA introduced into eukaryotic cells using standard methodology electroporation (Sambrook, J, E. F. Fritsch And T. Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition, Vols 1, 2, and 3, 1989). Cells in which LTVEC MA was introduced successfully, were selected by exposure to the selectivity of action of the agent. Because of breeding marker used in the modification cassette was neomycinphosphotransferase gene (neo) (Beck, et al., Gene, 19:327-36, 1982), cells that have absorbed the LTVEC MA, were selected in medium containing G418; cells that do not have LTVEC MA, was killed, while cells that have absorbed LTVEC MA, survived (Santerre, et al., Gene, 30:147-56, 1984).

Eukaryotic cells that have been successfully modified by targeting LTVEC ME in the locus MA identified using the method of quantitative PCR with TaqMan® (Lie and Petropoulos, Curr Opin Biotechnol, 9:43-8, 1998).

Finally, genetically modified ES cells used for the production of genetically modificirowan the x, in this case, with a knockout of the gene, mice standard blastocyst injection. Thus were created mice with knockout MA, i.e. mouse, which was deleterows gene MA.

As mice with knockout and wild-type mice (WT) were subjected to the action of inducing muscle atrophy conditions created by denervation of these mice, and the levels of atrophy compared. First was isolated sciatic nerve in the middle of the thigh of the right hind limb and cut in mice. Cutting the sciatic nerve leads to denervation and, during a 14-day period, to atrophy in the muscles of the hind limbs, in particular the anterior tibial and gastrocnemius muscles during a 14-day period. At 7 and 14 days after denervation animals were killed by inhalation of carbon dioxide. Then the anterior tibial muscle (TA) and gastrocnemius complex (GA) muscles were removed from the right (denervated) and the left (intact) of the lower extremities, weighed and frozen at some point in refrigerated liquid nitrogen isopentane. The degree of atrophy was assessed by comparing the weight of the muscle from denervated limbs with the weight of the muscles of nedarvinovskoy limbs.

Muscle atrophy was assessed after 7 and 14 days after cutting of the right sciatic nerve. Wet weight right denervated muscles compared with the raw weights of the left, nederevyannyh muscles. Comparison is possible by the right:the left are shown in table 2.

7 daysThe calf complexFront tibia
GenotypeThe sample sizeAverageSEThe sample sizeAverageSE
WT70,760,016110,680,033
NOC60,840,022110,800,015
14 daysThe calf complexFront tibia
The sample sizeAverageSEThe sample sizeAverageSE
WT50,550,02450,620,023
NOC50,800,01950,800,012

On days 7 and 14 of the muscles of mice with knockout found significantly (p&t; 0.001) and less atrophy than muscles from wild-type mice. The difference between mice with knockout and wild-type was greater at 14 days than at 7 days. Although mouse wild-type found ongoing atrophy between days 7 and 14, mice with knockout did not detect additional atrophy.

In General, we can conclude that the approach of creating LTVEC and their direct use as targeting vectors in combination with the screening MOA on the events of homologous recombination in ES cells creates a new method for constructing genetically modified loci, which is fast, inexpensive and represents a significant improvement over the tedious, time-consuming methods used previously. Thus, this approach opens the possibility of rapid large-scale functional genetic analysis of in vivo essentially any and all genes in the genome of an organism using only a fraction of the time and cost required for previous methods.

Although the foregoing invention has been described with some detail as illustrations and examples of professionals with expertise in this area will be clear that certain changes and modifications can be made in relation to the described invention without deviating from the essence or scope of the attached claims.

1. Pic is b genetic modification of interest to the endogenous gene or chromosomal locus in eukaryotic cells, providing:

a) obtaining a large cloned genomic fragment larger than 20 TPN containing interest DNA sequence;

b) using bacterial homologous recombination to genetically modify a large cloned genomic fragment of (a) to create a large targeting vector (LTVEC) for use in eukaryotic cells; the total size of the shoulders homology LTVEC is more than 20 TPN

c) introduction LTVEC (b) in eukaryotic cells for modifying endogenous gene or chromosomal locus in these cells by homologous recombination and

d) using a quantitative assay to detect modification of allele (MOA) in eukaryotic cells (C) to identify those eukaryotic cells in which the endogenous gene or chromosomal locus has been genetically modified.

2. The method according to claim 1, where a large cloned genomic fragment containing DNA sequences homologous interested endogenous gene or chromosomal locus.

3. The method according to claim 1 or 2, where the genetic modification of the endogenous gene or chromosomal locus includes the deletion of the coding sequence, gene segment, or regulatory element; change the coding sequence, gene segment, or re is estomago element; the insertion of a new coding sequence, gene segment, or regulatory element; creating a conditional allele or replacement of the coding sequence or segment of a gene of one species on homologous or ontological coding sequence from the same or another type.

4. The method according to claim 3, where the change in the coding sequence, gene segment, or regulatory element comprises a substitution, addition, or merge.

5. The method according to claim 4, where the merger involves epitope tag or a bifunctional protein.

6. The method according to any of the preceding paragraphs, where the quantitative analysis includes quantitative PCR, hybridization using fluorescence in situ (FISH), comparative genomic hybridization, isothermal amplification of DNA or quantitative hybridization to the immobilized probe.

7. The method according to any of the preceding paragraphs, where the eukaryotic cells are embryonic stem cells of a mammal.

8. The method according to claim 7, where embryonic stem cells are embryonic stem cells from mice, rats or other rodent.

9. The method according to any of the preceding paragraphs, where the endogenous gene or chromosomal locus is a gene or chromosomal locus of a mammal.

10. The method according to claim 9, where the endogenous gene or chromosome lococo what is a gene or chromosomal locus of the person.

11. The method according to claim 9, where the endogenous gene or chromosomal locus is a gene or chromosomal locus of the mouse, rat or other rodent.

12. The method according to any of the preceding paragraphs, where LTVEC able to accommodate large DNA fragments larger than 100 TPN

13. The method according to any one of claims 1 to 12, where approximately 1-5 μg large targeting vector (s) is introduced into approximately 1×107cells.

14. The method of genetic modification of interest to the endogenous gene or chromosomal locus in mouse embryonic stem cells, including:

a) obtaining a large cloned genomic fragment larger than 20 TPN, which contains the desired DNA sequence, where a large cloned DNA fragment homologous to the endogenous gene or chromosomal locus;

b) using bacterial homologous recombination to genetically modify a large cloned genomic fragment of (a) to create a large targeting vector for use in mouse embryonic stem cells, the total size of the shoulders more than 20 TPN, where genetic modification is a deletion of the coding sequence, gene segment, or regulatory element;

c) the introduction of a large targeting vector (b) in mouse embryonic stem cells for m the modification of the endogenous gene or chromosomal locus in these cells by homologous recombination and

d) using a quantitative assay to detect modification of allele (MOA) in mouse embryonic stem cells (C) to identify embryonic stem cells mouse in which the endogenous gene or chromosomal locus has been genetically modified, where quantitative analysis is quantitative PCR.

15. The method according to 14, where approximately 1-5 μg large targeting vector (s) is administered about 1×107cells.

16. Genetically modified endogenous gene or chromosomal locus for the application to obtain a genetically modified nonhuman organism where the gene or locus obtained by the method according to any one of claims 1 to 13.

17. Genetically modified eukaryotic cell to apply to obtain a genetically modified organism (not persons), where the cell receives a method according to any one of claims 1 to 13.

18. Genetically modified cell according to 17, which is an embryonic stem cell.

19. The use of genetically modified cells for 17 or 18 for receiving the body (not the person).

20. Genetically modified mouse embryonic stem cell to obtain a genetically modified mouse, where the cell receiving means 14.

21. The use of genetically modified mouse embryonaltoxikologie cells according to claim 20, for receiving the mouse.

22. The body (not people)that contains genetically modified endogenous gene or chromosomal locus obtained by the method according to any one of claims 1 to 13.

23. Mouse containing genetically modified endogenous gene or chromosomal locus obtained by the method according to 14.

24. The body (not human)derived from genetically modified eukaryotic cells by 17 or 18.

25. Mouse derived from genetically modified mouse embryonic stem cell according to claim 20.

26. The body (not people)that contains genetically modified interested endogenous gene or chromosomal locus obtained by the method, which involves stages:

a) obtaining a large cloned genomic fragment larger than 20 TPN containing the desired DNA sequence;

b) using bacterial homologous recombination to genetically modify a large cloned genomic fragment of (a) to create a large targeting vector (LTVEC) with the total size of the shoulders homology equal to more than 20 TPN, for use in embryonic stem cells;

(c) introducing the LTVEC (b) in embryonic stem cells for modifying endogenous gene or chromosomal locus in the cells by homologous recombination;

d) use quantitative the analysis to determine the modification of allele (MOA) in embryonic stem cells (C) to identify embryonic stem cells, in which this endogenous gene or chromosomal locus has been genetically modified;

e) introducing an embryonic stem cell (d) in the blastocyst and

f) introduction of a blastocyst (e) a surrogate parent female for gestation.

27. The body (not the man) p, which is the body of the mouse, rat or other rodent.

28. The body (not the man) p or 27, which is produced by the method according to any of claim 2 to 13.

29. Mouse containing genetically modified interested endogenous gene or chromosomal locus obtained by the method, which involves stages:

a) obtaining a large cloned genomic fragment, a larger 20 TPN, which contains the desired sequence DICK, where a large cloned fragment DICK homologous endogenous gene or chromosomal locus;

b) using bacterial homologous recombination to genetically modify a large cloned genomic fragment of (a) to create a large targeting vector for use in mouse embryonic stem cells, the total size of the shoulders homology large targeting vector is more than 20 TPN where this genetic modification is a deletion of the coding sequence, gene segment, or regulatory element;

c the introduction of large targeting vector (b) in mouse embryonic stem cells for modifying endogenous gene or chromosomal locus in these cells by homologous recombination and

d) using a quantitative assay to detect modification of allele (MOA) in mouse embryonic stem cells (C) identification of mouse embryonic stem cells in which this endogenous gene or chromosomal locus has been genetically modified, where quantitative analysis is quantitative PCR;

e) introduction mouse embryonic stem cell (d) in the blastocyst and

f) introduction of a blastocyst (e) a surrogate parent female for gestation.

30. Mouse on clause 29, which is obtained by the method according to claim 2-13.

31. The body (not people)that contains genetically modified endogenous gene or chromosomal locus obtained by the method, which involves stages:

a) obtaining a large cloned genomic fragment larger than 20 TPN containing interest DNA sequence;

b) using bacterial homologous recombination to genetically modify a large cloned genomic fragment of (a) to create a large targeting vector for use in eukaryotic cells (LTVEC); the total size of the shoulders homology large targeting vector is more than 20 TPN;

(c) introducing the LTVEC (b) in eukaryotic cells for genetic modification of the endogenous gene is or chromosomal locus in these cells by homologous recombination;

d) using a quantitative assay to detect modification of allele (MOA) in eukaryotic cells (C) for the identification of eukaryotic cells in which this endogenous gene or chromosomal locus has been genetically modified;

(e) removing the nucleus of eukaryotic cells (d);

f) injection engine (e) in the oocyte and

g) the introduction of the oocyte (f) a surrogate parent female for gestation.

32. The body (not the man) p, which is the body of the mouse, rat or other rodent.

33. The body (not the man) p or 32, which is obtained by the method according to claim 2-13.

34. The body (not people)that contains genetically modified endogenous gene or chromosomal locus obtained by the method, which involves stages:

a) obtaining a large cloned genomic fragment larger than 20 TPN containing the desired DNA sequence;

b) using bacterial homologous recombination to genetically modify a large cloned genomic fragment of (a) to create a large targeting vector for use in eukaryotic cells (LTVEC); the total size of the shoulders homology large targeting vector is more than 20 TPN;

(c) introducing the LTVEC (b) in eukaryotic cells for geneticallymodified endogenous gene or chromosomal locus in these cells by homologous recombination;

d) using a quantitative assay to detect modification of allele (MOA) in eukaryotic cells (C) for the identification of eukaryotic cells in which this endogenous gene or chromosomal locus has been genetically modified;

(e) merge eukaryotic cells (d) other eukaryotic cell and

(f) merged introduction eukaryotic cells (e) a surrogate parent female for gestation.

35. The body (not the man) 34, where the organism is a mouse, rat or other rodent.

36. The body (not people) in 34 or 35, which is obtained by the method according to any of claim 2 to 13.



 

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