JPH05507853A - Homologous recombination methods in animal and plant cells - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 20. Desired cells produced by the method of claim 11.

21. Desired cells produced by the method of claim 12.

22, a chimera containing a cell derived from the desired cell of claim 19 A non-human animal that is an animal or a transformed animal.

23, the animal and the desired cells belong to the same species and the inoculation is in chicken, mouse, rat Cat, hamster, rabbit, sheep, goat, fish, pig, cow or bull, or and a non-human primate according to claim 22. animals.

24. A chimera containing a cell derived from the desired cell of claim 20. A non-human animal that is an animal or a transformed animal.

25, the animal and the desired cells belong to the same species and the inoculation is in chicken, mouse, rat Cat, hamster, rabbit, sheep, goat, fish, pig, cow or bull, or and non-human primates according to claim 24. animals.

26, including cells derived from the desired cell of claim 21 or a line thereof. A non-human animal that is a chimeric animal or a transformed animal.

27, the animal and the desired cells belong to the same species and the inoculation is in chicken, mouse, rat Cat, hamster, rabbit, sheep, goat, fish, pig, cow or bull, or and non-human primates according to claim 26. animals.

28, containing a cell derived from the desired cell of claim 5 or a line thereof A non-fungal plant that is a chimeric plant or a transformed plant.

29. Introduction of desired non-selectable gene sequences into receptors requiring gene treatment. A method of gene treatment comprising: A. the desired non-selectable gene sequence; a DNA molecule containing a sequence of the desired gene sequence in a precursor cell of the receptor; the desired gene sequence sufficient to undergo homologous recombination with the intended gene sequence present in the An effective amount of a DNA molecule further containing two homologous regions flanking the gene sequence is B, allowing the introduction of said DNA molecule into said precursor cell; The introduced DNA molecule contains the predetermined gene sequence of the genome of the precursor cell. The desired non-selectable gene sequence is obtained by subjecting it to homologous recombination. Create a desired cell in which the receptor is inserted into the predetermined gene sequence. The presence or expression of the introduced gene sequence in cells constitutes the gene therapy. A method consisting of things.

30. The method of claim 29, wherein said receptor is a non-fungal plant.

31. The method of claim 29, wherein said receptor is an animal.

32. The animal is a chicken, mouse, rat, hamster, rabbit, sheep, or goat. , fish, pigs, cows or bulls, non-human primates, and humans? 32. The method of claim 31, wherein the method is selected from:

33. The method of claim 32, wherein the animal is a human.

34. A desired non-selectable gene inserted within a predetermined gene sequence in the genome of a cell. A method for obtaining a desired animal cell or non-fungal plant cell containing a genetic sequence, the method comprising: A, Precursor cells were cultured under non-selective culture conditions or under selective culture conditions of 1) following i) and ff) with a DNA molecule containing the gene sequence of [i) the desired a non-selectable gene sequence (wherein the DNA molecule is a precursor to the desired gene sequence) sufficient to cause homologous recombination of the genome of a somatic cell with the predetermined gene sequence. further containing two homologous regions flanking the desired gene sequence), and ti) a selectable gene sequence, the presence or expression of which in said progenitor cell; selecting the cells by culturing the cells under the first selective culture conditions; and its presence or expression in the precursor cells is determined by a second selective culture. Gene sequences that can be negatively selected by culturing the cells under Column] B. Enables the introduction of the DNA molecule into the precursor cell, C9. A molecule undergoes homologous recombination with the predetermined gene sequence of the genome of the progenitor cell. by causing the desired non-selectable gene sequence to become the predetermined gene sequence. D. Under the first selective culture conditions, the desired cells are inserted into a column. the cells are cultured and then allowed to undergo intrachromosomal recombination under non-selective culture conditions. by incubating the cells under the second selective culture conditions. and collecting the desired cells.

35, said cell is HPRT enzyme deficient and said selectable gene sequence is active PRT enzyme is expressed, and the first selective culture conditions are such that the PRT enzyme is present in the cells in order to grow. Incubating the cells under conditions requiring the presence of active HPRT enzyme The second selective culture conditions are such that the second selective culture conditions require activity within the cells in order to grow. Incubating the cells under conditions requiring the absence of active HPRT enzymes. 35. The method of claim 34, comprising:

36, said cell is APRT enzyme deficient and said selectable gene sequence is active A PRT enzyme is expressed, and the first selective culture conditions are such that the PRT enzyme is present in the cells in order to grow. Incubating the cells under conditions requiring the presence of active APRT enzyme The second selective culture conditions are such that the second selective culture conditions require activity within the cells in order to grow. Incubating the cells under conditions requiring the absence of active APRT enzyme. 35. The method of claim 34, comprising:

37, said cell is TK enzyme deficient and said selectable gene sequence is an active TK enzyme The first selective culture conditions require active T in the cells to grow. by incubating the cells under conditions requiring the presence of the K enzyme. and the second selective culture conditions require active TK enzyme in the cells for growth. consisting of incubating said cells under conditions requiring the absence of The method of claim 34.

Specification Cross-reference of applications related to homologous recombination in animal cells and plant cells This application is based on U.S. Patent Application No. 071537458 (filed in the Philippines on June 14, 1990). This is a partial continuation application.

field of invention The present invention relates to recombinant DNA technology, and more specifically to chimeric animals and plants or transformation. This invention relates to a method for modifying endogenous genes in motile plants. Furthermore, the present invention provides that the method The present method in relation to animals/plants produced by application and in medicine and agriculture. Regarding the use of. This invention was supported by (US) Government funds.

The (US) Government has certain rights in this invention.

Background of the invention ■, Chimekrates and transformed animals Recent advances in recombinant DNA and gene technology have made it possible to transfer desired gene sequences into recipient animals. This made it possible to express this in recipient animals. A method like this gene sequences not normally present in unmodified animals or in unmodified animals. Animals have been engineered to carry genetic sequences that do not occur naturally.

These techniques also allow the creation of animals with altered expression patterns of naturally occurring gene sequences. It has also been used to create

Animals created by using these methods are “chimeric” or Known as "transgenic" animals. “Chimera” animals In this case, the cells that contain and express the introduced gene sequence are those of that animal. The rest of the cells remain unaltered. chimeric animals were introduced The ability to transmit a gene sequence to its offspring depends on whether the introduced gene sequence is present in the animal's life. Depends on whether it is present in the reproductive cells. Therefore, the desired gene sequence can be There are only certain types of chimeric animals that can be passed on to offspring.

In contrast, all cells of a “transformed” animal contain the introduced gene sequence. have. Therefore, transformed animals transmit the introduced gene sequence to their offspring. can be reached.

■ Creation of transformed animals: Microinjection method Transformation The most widely used method for creating the Involves injection of NA molecules (Brinster, R, L, et al. Ce1127: 223 (1981): Co5tantini, F. et al., Nature 29 4:92 (1981):■arbers, K. et al., Nature 293: 54O (1981); fagner, E, F, et al., Proc, Natl, Acad, Sc i, (U, S, ^) 78:5016 (1981); @Gordon, J.? , et al., Pr. oc, Natl, Acad, Sci, (U, S, A,) 73: 1260 (1976)).

The introduced gene sequence is a self-replicating plasmid or virus of some kind. (Jaenisch, R., 5science). , 240:1468-1474 (1988)). In fact, the presence of vector DNA is often found to be undesirable (Hammer, R.E., et al. , 5science 235:53 (1987); Chada, K. et al. Nature 319:6W5 (1986); K.

11ias, G et al., Ce1146:89 (1986); 5hani, M, , Mo1ec, Ce11. Biol, 6:26Q4 (1986) ;　Cha da, K. et al., Nature 314:377 (1985); Town es, T et al., EMBOJ, 4:1715 (1985j).

After the DNA molecules are injected into the recipient fertilized egg, they recombine with each other to form a head-to-tail concatenation. It is thought to form a temer (chain-like body). This kind of concatemer is the egg Occurs at the double-stranded DNA cleavage site at any site in the chromosome, and constructs at such sites It has been proposed that catemers are inserted and incorporated (Brinster, R. L, et al., Proc, Natl^cad, Sci, (It, S, A, ) 82:443g (1985)). Therefore, the injected DNA molecules are fertilized can be incorporated into several locations within the egg chromosome, but is most often observed There is only one insertion site (Jaenisch, R., 5science, 2 40:1468-1474 (1988): Meade, hll et al., U.S. Pat. No. 4,873,316).

Once the DNA molecule is injected into the fertilized egg cell, the cell is implanted into the uterus of the recipient female. develop in animals. All of the animal's cells are derived from the transplanted fertilized egg. , all of the resulting animal cells (including germline cells) contain the introduced gene. It should contain the sequence. Before the introduced gene sequence integrates with the cell's genome If the first cell division occurs in (which happens about 30% of the time), the resulting dynamics Things will become chimeric animals.

By breeding or inbreeding such animals, heterozygous and homozygous It has become possible to create type-conjugated transgenic animals. Such transformation behavior Despite the unpredictability of object formation, these animals are generally stable. It has been shown that it is possible to produce offspring that retain and express the introduced gene sequence. I know.

Microinjection results in integration of the injected DNA into the genome of the fertilized egg, but The process involves disruption and modification of the nucleotide sequence of the egg chromosome at the insertion site. Therefore, the function of the endogenous egg gene at the site where the injected DNA is inserted may be altered or destroyed. It has been observed that this results in disruption or deletion. Furthermore, the inserted DNA Substantial modifications (deletions, duplications, transpositions, and transpositions) of endogenous egg sequences adjacent to has been observed (Mahon, K., A. et al., Proc. Natl., A. cad, Sci, (U, S, A,) 85:11U5 (1988) ; Covarrubias, Y., et al., Proc., Natl., Acad. Sci, (U, S, A,) 83:6020 (P986); M ark, Y, et al.

Col1d Spr, Harb, Symp, Quant, Biol, 5 0+453 (1985)). In fact, lethal mutations or significant morphological abnormalities has been observed (Jaenisch, R., 5science 240:1 468-1474 (1988); First, N., L. et al., Amer , Meat Sci, As5n, 39th Reciprocal Me at bonf, 39:41 (198 6)).

Significantly, even if the desired genetic sequence of the microinjected DNA molecule is Even if the desired gene sequence is naturally found in the genome of It has been observed that Brin rarely occurs at the natural genetic site of ster, R, L et al., Proc, Natl, Acad, Sci, ( U, S, A, ) 86: 701117-7091 (198X)). Sara to the desired Introduction of a gene sequence generally does not alter the originally existing egg gene sequence.

Determining in advance the site in the fertilized egg genome where the injected DNA will ultimately be integrated. expression of the desired gene sequence in the animal is organ- or tissue-specific. It is possible to control the expression of a desired gene sequence so that it occurs in a Westphal, H., FASEB J, 3:117 (1989): Jaenisch, R,,5science 240:1S6g-1474(1 98 8)).

Mice have the highest success rate for creating transgenic animals. The DNA was injected and transplanted into a female. Approximately 25% of fertilized mouse eggs will become transgenic animals. As of now, Success rates are lower in herons, sheep, cattle and pigs (Jaenisch, R, 5science 240:1468-1474 (1988): Il lammer, R.E., et al., J. Animal, Sci. 63:269 i1986): HamIIler.

R, E, et al. Nature 315:680 (1985): Wagner , T., E. et al., Theriogenology 21:Q9 (1984)) .

This low success rate may be due to the fact that the mouse is better understood as a genetic system, This is a reflection of the difficulty in visualizing the male pronucleus in fertilized eggs of many domestic animals. (Wagner, T., E. et al., Theriogenology 21:2 9 (1984)).

Therefore, the creation of transgenic animals by microinjection of DNA involves at least two main approaches. It has important drawbacks. First, this method can only be performed during the single-cell stage of animal life. No. Second, this method requires disruption of the natural sequence of DNA and therefore often mutagenic or teratogenic (Gridley, T. et al., Tr. ends Genet.

3:162 (1987)).

■ Creation of chimeric animals and transformed animals, recombinant virus methods, and recombinant retrovirus methods Lovirus method Chimeric and transformed animals can be produced using recombinant virus technology or recombinant retroviruses. It can also be created using standard technology. This technique involves gene sequencing during the multicellular stage. introduced into animals. In such methods, the desired genetic sequence is transferred to a virus or Introduced into torovirus. Cells infected with this virus are infected with the introduced gene. Get array. This virus or retrovirus is present in all cells of the animal. This method produces transformed animals. but the virus Chimeric animals can be created if the virus infects only some of the animal's cells. It becomes.

Expression of non-replicating DNA in viral or retroviral transgenic animal production methods A general advantage over methods involving microinjection is that genetic manipulation does not need to be performed at the single-cell stage. That means no. Furthermore, infection is extremely difficult to introduce DNA into the desired cells. This is an effective method.

Recombinant retroviral methods for creating chimeric or transformed animals are Toroviruses integrate into the host genome in a precise manner (although multiple insertions may occur). (Nono) In general, the advantage is that there is only one integrated retrovirus. Has a point. Relocation of the host chromosome at the integration site is generally limited to small deletions. (Jaenisch, R, 5science 240:1468-14 74 (1988); Varmus, H., “RNA@Tumor V iruses'' (Weiss, R. et al., eds., Cold Spring Harbor Press) (Cold Spring Harbor, New York), pp. 369-512 (1982). ). However, this method is as mutagenic as microinjection. be.

Chimeric animals can be used, for example, for viruses that can infect the cells of a host animal (bovine papilloma mouse). It is created by inserting the desired gene into a virus, polyoma, etc.). Ta. Once infected, the virus can be maintained in infected cells as extrachromosomal episomes. (Elbrecht, A. et al., 1Jolec, Ce11.Biol, 7:1276 (1987): Lacey, M. et al. @Nature 32 2: 609 (1986): Leopold, P. et al. Ce1151:885 ( 1987)). Although this method reduces the mutagenicity of chimera/transgenic animal formation, , this decrease is caused by decreased germline stability and increased tumorigenicity. Koru.

Pluripotent embryonic stem cells (referred to as “ES” cells) are used during the early post-implantation stages of embryogenesis. cells that can be obtained from embryos. These cells cannot be grown in culture. It can be differentiated in vitro, and if transplanted as a tumor into mice, It can also be differentiated in vivo. ES cells have a normal cutting pattern (Evans , M., J. et al.

Nature 292:154-156 (1981); Martin, G. , R, et al., Proc, Natl, Acad, Sc weir A (U, S, A,) 78 Near 634-7638 (1981)).

When injected into the blastula of a developing embryo, ES cells proliferate, differentiate, and thus It will bring about the creation of la animals. ES cells are the somatic cell system of such chimeric animals. can colonize both the column and germline (Robertson, E. , et al., Co1d Spring Harb, Conf, Ce1l Pro lif, lo: 647-663 (1983); Bradley, A. et al. Nat buoyancy ■@309:255 -256 (1984): Bradley, A. et al., Curr, Top , Devel, Biol, 20:357-371 (1X86): Wa gner, E.

F, et al., Cold Spring Harb, Symp, Quant, B iol, 50:691-700 (1985); these ■■■ (all of which are incorporated herein by reference).

In this method, ES cells are cultured in vitro, and cells containing the gene sequence of interest are grown. infection with viral or retroviral vectors. retro virus vector Chimeric animals created using vectors are germ cells in which the introduced gene sequence has been deleted. or alternatively, generate progeny cells that contain the introduced sequences but are capable of expressing the introduced sequences. It is known that they have germ cells lacking the ability to produce (Evas, M. , J, et al., ColdSpring Harb, Symp, Quant, BioL 50:685-689 (1985): 5tevart, CCL , et al. EMBO1 4:3701-3709 (1985); Robertson, L., et al., N. (1986); these documents constitute a part of this specification).

Since ES cells can be grown in vitro, somatic cell genetics techniques can be used to It is possible to manipulate cells. Therefore, (e.g. hypoxanthine phospho (e.g. in the hprt gene, which encodes ribbon letransferase) It is possible to select ES cells that retain mutations (Booper, M et al. Nature 326:292-295 (1987); Kuehhn, M. R et al., Nature 326:295-298 (1987)). Then this ■ By using cells selected in such a way that they cannot express active HPRT enzymes. Chimeric or transgenic mice can therefore be created (e.g. animal models of diseases (such as Leanyu-Nyhan syndrome, which is characterized by HPRT deficiency). (Doetschman, T. et al., Proc. Natl., A. cad, Sci, (U, S, A,) 85:8T83-8587( 19 88)).

As mentioned above, the texture (with germline cells containing the introduced gene sequence) It is possible to create transgenic animals from La animals by inbreeding.

The above-described method requires that the desired This makes it possible to screen for genetic modifications. However, this One drawback of these methods is that the site or nature of vector integration cannot be controlled. That's a good thing.

■ Production of chimeric animals and transformed animals Plasmid method The above chimeric animals and There are inherent shortcomings in the methods used to create transgenic and transgenic animals, making it difficult for researchers to We have attempted to identify other ways in which the .

For example, Gossl, er, A et al. Use of a plasmid vector modified to contain the gene (nptII gene) describes the transfection of cultured ES cells using nptI The presence of the I gene makes ES cells infected with this plasmid resistant to the antibiotic G418. conferred resistance to (Gossler, A., et al., Proc., Natl. Acad, Sci, (U, S, ^,) 83:9065-9069 (P9 86) ;This sentence (Contributions are incorporated herein by reference). received the above plasmid and directed against G418. Chimeric animals that have become resistant to this vector have integrated this vector into their chromosomes. I understand. Takahashi, Y., et al. used plasmids to develop avian Describes the creation of chimeric mouse cells that express the crystallin gene. (Development 102:258-269 (1988); this document constitutes a part of this specification). This avian gene contains the nptII gene integrated into a plasmid. The obtained chimeric animal is capable of expressing the above avian gene. I found out.

■ Introduction of gene sequence into somatic cells Various cell lines have been created by introducing DNA into somatic cells. Hara o Mat, Cell Genet, 5:1031-1044 (1979)). F Olger et al. reported that thymidinequina was microinjected into the nucleus of cultured mammalian cells. The fate of the tkase gene (tk gene) was investigated.

The recipient cell has integrated concatemers at one or several locations in its genome. was found to contain 1 to 100 copies of the introduced gene sequence (Fo lger.

KR et al., Molec, Ce11. Biol, 2:1372-1387 (198 2)). Introducing the RNA polymerase gene into Syrian hamster cells via DNA. It has also been reported that the introduction of 11. Biol, 2:666-673 (1982)).

Confers neomycin resistance and guanosine phosphotransferase activity to the host Transfecting Chinese hamster ovary cells with a plasmid that New cell lines have been created (Robson, C, N et al., Mutat , Res, 163:201-208 (1986)).

■ Chimecula or transformed animals In Omi, the field of plant cell genetics and gene technology has made significant progress. many from single cells by using protoplast regeneration techniques for plants of the genus Plants can be regenerated (Friedt, et al., Prog, Botan y 49:192-215 (1987); Brunold, C et al., Mo1 ec, Gen, Genet, 208:469-473 (1987); Durand, i, et al. Plant 5ci 62:263-272 (1989); these documents constitute a part of this specification. ).

Several methods are available for delivering and expressing foreign genes into plant cells. et al., J. Bacteriol, 166:8g-94 (1986): C zako, M, et al., Plant Mo1. BiolA 6 + 101- 109 (19 86); Jones, J., D., G. et al., EMBOJ, 4:2411 -2418 (1985); 5hahin, E, A, et al. Theor, Appl, G enet, 73:164-169 (1986)). The frequency of transformation is about 70% depending on the type of plant used (Friedt, et al., Pro g, Botany 49:192-215 (1987)).

Plant viruses are also used for foreign gene delivery to and expression of foreign genes in plants. It has been explored as a vector. Cauliflower mosaic virus (Brisso) N, N et al., Nature 310:511-514 (1984)) for this purpose. It has become particularly useful for (Shah.

D, ll et al., 5science 233:47g-481 (1986): Shevmaker, C.K., et al., Virol, 140F281-288 ( 198 5)). Vectors have also been created from derivatives of RNA viruses (French et al. h, R, et al., 5science 231:1294-1297 (1986)).

Microinjection techniques (Crossvay, A et al., Molec, Gen, Ge net, 202:179-185 (1986): Po us, 1. et al., Molec, Gen, Genet, 199: 169 -177 (1985)), direct transfer of gene sequences into plant cells was achieved. It is. Koji using transformation with a plasmid capable of site-specific recombination. Gene sequences have been introduced into the fungal genus (May, G., S., J. Ce11 Biol, 109:2267-2274 (1989); this document constitutes a part of this specification).

Electroporation is a method of introducing DNA into plant cells. (Fromva, M, E et al., Proc, Natl, Acad, Sci, (U, S, A,) 82: T824-5828 (1 985); Fromm, M, E et al., Nature 319 Near 9l -793 (1986): Morikawa, H et al., G■shi■@41:12 1-1 24 (1986); Langridge, f., H.R., et al., Theo r, Appl, Genet, 67:443-455i1984)).

Significant genetic mutations can be created in plant cells using transposable elements ( Saedler, K. et al., EMBOJ, 4:585-590 (1985 ) ; Peterson, P, A, , BioE Tang Tang≠Katou@3:19 9-204 (1 985))c Such factors cause chromosomal rearrangements, insertions, duplications, deletions, etc. It can happen. Chimeric plants can be generated from such cells by using the techniques described above. can be played.

A major flaw in current methods for plant genetic engineering is the selection of the desired recombinant cells. (Bruaold, C. et al., Molec, Gen. , Genet, 208:469-473 (1987)j.

In an attempt to address this defect, kammycin resistance and nitrate reductase deficiency DNA loss is used as a selectable marker (Brunold, C. , et al., Molec, GenGenet, 208+469-473 (198 7)).

■、Conclusion Application of the techniques described above has led to the creation of types of plants and animals that cannot be created using classical genetics. have the potential to create. For example, human diseases (e.g. AIDS, diabetes, animals affected by diseases such as cancer, which may be useful in elucidating treatments for such diseases. You can create animals. Chimeric and transformed animals and plants are natural genetic It has important uses as a probe (examination tool) for child expression. Livestock using these techniques and when applied to food crops, these techniques can provide improved food, fiber, etc. has the potential to

Despite the success of the above-mentioned techniques, less mutagenic and specific chromosomal locations A method for creating chimeric animals and plants that enables clear, specific, and precise manipulation of inserted gene sequences. Alternatively, a method for producing transformed animals and plants is strongly desired.

Brief description of the drawing FIG. 1 depicts the use of replacement and insertion vectors in gene targeting methods.

Figure IA is a schematic representation of the use of replacement genes in gene targeting methods; IB is the use of insertion vectors to create minute mutations in the desired gene sequence. Represents business.

Figure 2 shows a schematic diagram of a DNA molecule with a heterologous structural region located at the intended insertion site. It is an expression. Figure 2A represents a construct with a 2kb heterologous region. Figure 2B shows the difference Represents a construct with a 26 base long heterologous region linearized in the middle of the seed structure region .

Figure 20 shows the heterologous structural region located within the homologous region at which recombination is desired. represents the associated construct. In Figure 20, the normal amHI site of the vector is Nhe The vector's normal EcoRI site has been converted to a BamH1 site. has been converted. This vector is linearized with XhoI.

Figure 3 shows the mechanism by which ``anthropomorphic'' genes can be introduced into chromosomal gene sequences in a one-step process. This is a schematic representation of the structure.

Figure 4 shows how a large gene is placed under the transcriptional control of a heterologous promoter. (e.g., so that human genes are placed under the control of mouse genes) Schematic representation of mechanisms that can be introduced into chromosomal gene sequences. The first stage is additional The second stage is a substitutional event. Figure 4A shows the first stage of this process; Figure 4B shows the second stage of this process. If desired, all mouse code exons are included. A repair recombination event can be configured such that all are removed.

Figure 5 shows the positive selection/negative selection “cassette” for introducing small mutations into chromosomes. This is a schematic representation of the use of ``cut''.

Figure 6 shows the sequence of small or large desired gene sequences in the cell genome. This is a schematic representation of the multi-step method (Figures 6A to 6E) for introduction into the domain. Ru.

This diagram shows overlapping clones for building large loci. 2 illustrates a vector that can facilitate sequential addition of molecules. Each stage is selectable. Ru.

Repeat steps 4 and 5 as many times as necessary to prepare the inserts in HAT medium. by selecting for repair in medium supplemented with 6-thioguanine. Further additions can be made. If necessary, at the other end of this locus This approach can also be achieved.

This is a schematic representation of the vector used in this experiment.

Figure 8 shows the expected hprt gene after homologous recombination of the IV6.8 vector. Represents a structure. HR is a fragment of the expected size indicative of this homologous recombination event. .

End, D is the endogenous fragment that is duplicated by this recombination event. End is , the expected neighbors detected by the partial cDNA probes used in these experiments. It is a fragment.

Figure 9 shows the reversion of homologous recombinants generated by the insertion vector.

Figure 10 shows the use of polyA selection as a means to select for homologous recombination events. represent.

FIG. 11 shows the method of the present invention for introducing an insert into the sequence of a desired gene in a cell. Represents use. Figure 11A is a schematic of the c-src locus showing relevant restriction sites. It is a diagram (E = EcoRI; N = NcoI; X = XhoI: H = HindI: B=BamH1; Nh=Nhel). Figure 11B is c-s 5rc14 vector used to introduce mutations into the rc locus However, Figure 11c depicts the subtle mutations introduced by the use of this vector.

Figure 12 illustrates the use of the present invention to introduce substitutions into the sequence of a desired gene in a cell. Represents business. Figure 12A is a schematic of the c-src locus showing the relevant restriction sites. (E-EcoRI; N=NcoI: X=Xhol; H=Hind B=Ba+*HI:Nh=Nhel). Figure 12B shows c-src inheritance Figure 12 represents the 5rc33 vector used to introduce mutations into the child locus. C represents a minute mutation introduced/introduced by use of this vector.

Figure 13 depicts a comparison of targeted and random recombination events. random recombination In this event, concatemers can excise the duplicate, but one copy of the vector remains in the genome. must remain in In contrast, a targeted recombination event involves all steps to remove the desired sequence. all sequences are excised from the genome.

Summary of the invention The present invention provides a method for preparing a desired protein containing a predefined specific desired modification in its genome. Methods of obtaining animal cells or non-fungal plant cells are provided. Moreover, the present invention Relates to non-human animals and plants that can be produced from cells. Furthermore, the present invention Regarding the use of plants and animals other than humans and their descendants in research, medicine, and agriculture. Ru.

Specifically, the present invention provides a method for detecting a desired gene sequence inserted within a predetermined gene sequence of the genome of a cell. Obtain the desired animal or non-fungal plant cell containing the non-selectable gene sequence of A method of A, a DNA molecule containing a desired non-selectable gene sequence and a desired non-selectable gene sequence; undergoes homologous recombination with the expected gene sequence in the genome of the progenitor cell. further contains two homologous regions adjacent to the desired gene sequence sufficient to B. incubating the precursor cell with the DNA molecule; C6 introduces the introduced DNA molecules into the precursor cell genome. A desired selection defect is achieved by homologous recombination with a planned gene sequence. create a desired cell in which a possible gene sequence is inserted into the intended gene sequence; D. Collecting desired cells; Provides a method consisting of:

Furthermore, the present invention provides that the DNA molecule contains a detectable marker gene sequence and/or Alternatively, by electroporating precursor cells and DNA molecules. DNA molecules are introduced into progenitor cells (particularly in step B, progenitor cells are detected). simultaneously with a second DNA molecule containing a marker gene sequence that can be roporation).

Furthermore, the present invention provides that the desired cells are plant cells other than fungi, somatic animal cells (particularly chicken , mouse, rat, hamster, rabbit, sheep, goat, fish, pig, cow or selected from the group consisting of bulls, non-human primates, and humans); Competent animal cells (especially chicken, mouse, rat, hamster, rabbit, sheep, From the group consisting of goats, fish, pigs, cows or bulls, and non-human primates (selected ones)). The present invention allows pluripotent cells to Includes embodiments that are stem cells.

Further, the present invention provides that the desired gene sequence is essentially the same as the predetermined gene sequence of the precursor cell. homologous and/or the desired gene sequence is similar to the predetermined sequence of the progenitor cell. Embodiments of the above-described methods that are analogues (particularly human analogues) are included.

In addition, the present invention provides that a desired gene sequence is used for hormones, immunoglobulins, receptors. receptor molecules, ligands of receptor molecules, and enzymes. It also includes embodiments encoding proteins.

The present invention also includes an introduced recombinant DNA molecule containing the desired gene sequence. a non-fungal plant cell, the desired gene sequence of which contains a predetermined gene sequence of the cell's genome; The desired gene sequence has enough homologous regions to undergo homologous recombination with the gene sequence. Also includes adjacent plant cells.

The present invention also includes an introduced recombinant DNA molecule containing the desired gene sequence. A non-human animal cell, the desired gene sequence of which is present in the genome of the cell. The desired gene sequence has enough homologous regions to undergo homologous recombination with the desired gene sequence. It also includes animal cells that are adjacent to the column.

The invention also encompasses desired cells produced by any of the methods described above.

The present invention also includes cells derived from the desired cells or their progeny. It also includes non-human animals that are chimeric or transformed animals, and in particular humans other animals and the desired cells belong to the same species, and the species is chicken, mouse, rat, etc. animals, hamsters, rabbits, sheep, goats, fish, pigs, cows or bulls, and and non-human primates.

The present invention also provides textured cells containing cells derived from the desired non-fungal plant cells. It also includes non-fungal plants that are plants or transformed plants.

The present invention also provides a method for providing a desired non-selectable gene sequence to a recipient in need of gene therapy. A method of gene therapy comprising introducing A. a desired unselectable gene; A DNA molecule containing a genetic sequence, and in which the desired genetic sequence is present in the recipient. sufficient to undergo homologous recombination with the intended gene sequence present in the progenitor cell. , a DNA molecule that further contains two homologous regions flanking the desired gene. amount of C9 to the recipient, B. allowing the introduction of said DNA molecule into the precursor cell; By causing the introduced DNA molecule to undergo homologous recombination with the precursor cell , the desired non-selectable gene sequence is inserted into the predetermined gene sequence. create cells of This means that the presence or expression of the introduced gene sequence in the cells of the recipient is inheritable. Includes methods of structuring child therapy.

Specifically, the invention provides that the recipient is a non-fungal plant or a human or non-human animal. (Specifically chicken, mouse, rat, hamster, rabbit, sheep, goat, Selected from the group consisting of fish, pigs, cows or bulls, non-human primates and humans. Embodiments of the above-described method include embodiments of the above-described method in which a non-human animal is selected.

The present invention also provides for the insertion of a desired gene sequence into a predetermined gene sequence of the genome of the cell. a desired animal cell or non-fungal plant cell containing a non-selectable gene sequence of A method of obtaining A, Progenitor cells were subjected to the following i) under non-selective culture conditions or under first selective culture conditions: and if) a DNA molecule containing the gene sequence of [i) the desired selection. unselectable gene sequence (where the above DNA molecule is further sufficient to cause homologous recombination with the intended gene sequence in the genome of the progenitor cell, containing two homologous regions flanking the desired gene sequence), and it) a selectable gene sequence, the presence or expression of which in a progenitor cell; can be selected by culturing the cells under first selective culture conditions. and its presence or expression in the progenitor cells is determined under second selective culture conditions. Gene sequences that can be negatively selected by culturing the cells with]B , allows the introduction of the DNA molecule into the precursor cell, and allows the introduction of the DNA molecule into the precursor cell. Allowing the offspring to undergo homologous recombination with the desired gene sequence in the genome of the progenitor cell. The desired non-selectable gene sequence is inserted into the predetermined gene sequence by D. Cultivate the cells under the first selective culture conditions; The cells are allowed to undergo intrachromosomal recombination under non-selective culture conditions, followed by a second selection. Harvest the desired cells by incubating the cells under selective culture conditions Provide a method consisting of:

The present invention provides that the cells are HPRT, APRT or TK enzyme deficient and Possible gene sequences express active HPRT, APRT or TK enzymes, The first selective culture conditions are active HPRT, APRT or TK for growth. Incubating cells under conditions that require the enzyme to be present within the cell and the second selective culture condition is HPRT active for growth.

Cells are grown under conditions that require the absence of APRT or TK enzymes within the cells. It also includes embodiments consisting of incubating.

Description of preferred embodiments The present invention relates to a method of introducing DNA into the genome of a recipient animal or plant cell. Chimera In order to create animals or transgenic rabbits, sheep, goats, fish, pigs, cows, and non-humans The use of natural DNA to introduce such NA into germline cells of primates) be able to. You should also create chimeric or transformed plants (using natural DNA can also be introduced into plant cells and then manipulated.

or use nature to modify the somatic cells of animals (including humans) or plants. can be done. can be operated by applying the methods disclosed herein. All multicellular higher (i.e., non-fungal or non-yeast) plants and plant cells Contains things.

■, homologous recombination The present invention provides methods for introducing desired gene sequences into animal and plant cells. Therefore to create chimeric or transgenic animals and plants with defined and specific genetic modifications. can be achieved.

In order to fully understand the present invention, it is desirable to understand the process of homologous recombination. (latson, J, D, “Mo1ecular Biology of the Gene”, 3rd edition, W. A. Benjamin, Inc. Menlo Park, Calif. (1977), which document is incorporated herein by reference. ).

Briefly, homologous recombination is a well-studied natural cellular process, and this have the same or essentially similar sequence (i.e. “homologous”) ”) The cleavage of two nucleic acid molecules and the joining of the two molecules occur, resulting in the formation of the original One region of each molecule is linked to a region of the other molecule that was originally present. (Sedivy, J. M., Bio-TechnoL6: 11 92-1196 (1988), which is incorporated herein by reference).

Homologous recombination is therefore a sequence-specific process that allows cells to A DNA "region" of one DNA molecule can be transferred to another DNA molecule. Book As used herein, a “region” of DNA refers generally to any nucleic acid molecule. To be. This region can be of any length, from a single base to a considerable length of a chromosomal fragment. It may be of the same name.

For homologous recombination to occur between two DNA molecules, those molecules must “ Homologous regions” must be retained. Such homologous regions are few (both 2 and 3). Must be base pair length. one DNA molecule in a region of a second DNA molecule In contrast, it contains a region consisting of sufficiently similar sequences that homologous recombination can occur. In this case, these two DNA molecules are said to possess such "regions of homology".

Recombination is catalyzed by enzymes that occur naturally in both prokaryotic and eukaryotic cells.

Transfer of a region of DNA can be imagined to occur through a multistep process.

If one of the two participating molecules is a circular molecule, the recombination event described above This results in the incorporation of the cyclic molecule into the participating molecule.

Importantly, a given region has multiple homologous regions (which may be the same but different). (preferably) are adjacent, two recombination events occur. Therefore, exchange of DNA regions can occur between two DNA molecules. Recombination is “reciprocal” recombination can cause the crossing of DNA regions between two recombinant DNA molecules. exchange may result. Or, recombination is “non-reciprocal” (also called “gene conversion”). DNA molecules that are both recombinant and have the same nucleotide sequence Sometimes it is. Regarding the size or sequence of the region exchanged in two-event recombination exchange. There are no restrictions.

By treating the introduced DNA with a substance that stimulates recombination, two DNA The frequency of recombination between molecules can be increased. Examples of such substances include Includes chillpsoralen, UV light, etc.

■Creation of chimeric organisms and transgenic animals: gene targeting, clearly defined specificity One way to create animals with major genetic modifications is to In a fusion of ploid human fibroblasts and tetraploid murine erythroleukemia cells, the transduced matrices Homologous recombination is used to control the integration site of the marker gene sequence. (Sa+1thies, O, et al., Nature 317:230-234(1 985)).

This method was further exploited by Thomas, K. R., and co-workers. and they pre-selected desired cells in ES cells to create transgenic animals. A general method for making targeted mutations in gene sequences (this is called “gene targeting”). Mansour, S, L, et al. , Nature 336:348-352 (198g); Capecc■ i, M, Ro, Trends Genet, 5: 7O-76 (19 89) ; Capecchi, M., R. et al., Cure Shibut@Commun cation s in MoLular Biology-, Capecchi, M. 11. ed., Cold Spring Harbor Press.

Cold Spring Harbor, NY (1989), pp. 45-52: (These documents constitute a part of this specification).

The gene targeting method involves insertion of the nptII gene into the β2-microglobulin locus. CKoll used to create chimeric mice and transgenic mice er et al., B.

■, Ra, Proc, Natl, Acad-Sci, (U, S, A, ) 86: 8932-8935 (1989): Zij Gekikarasoni = v, et al. Nature 342:435-43 (1989): Zijlstra, M. et al., Nat. ure 344: 742-746 (1989); Debhiaba et al. Nat rure 345 Near 8-80 (1990)). Similar experiments revealed that nptI Chimeric animals and forms with c-abl gene disrupted by insertion of I gene It has become possible to create transgenic animals (Schwartzberg, P., L. , et al., 5science 246:799-803 (1989)). This technology is By inserting the 1■ gene! ” Chimeric mice with two λ genes disrupted It is also used to create (Joyner, A. L. et al., Natur E 338:153-155 (1989)).

Gene targeting methods have also been used to correct hprt defects in hprt-ES cell lines. is also used. Chimeric animals were created using cells with this defect corrected. Ta. Significantly, all of the corrected cells contained regions adjacent to the hprt locus. showed significant destruction of the area and all of the cells tested were used to correct the above defects. and containing at least one copy of the vector integrated into the hprt gene locus. Got it (Thompson, S. et al., Ce1156:313-321 (19 89); Koller, B., Ill., et al., Pr. Natl. ^cad, Sci, (U, S, A,) 86:8927-8931 (1 989)).

In order to use the “gene targeting method,” the gene of interest must be cloned in advance. and its intron-exon boundaries must be determined.

This method involves injecting a marker gene (i.e. nptII gene). Therefore, the use of this gene targeting method This results in significant disruption of the gene of interest.

Recently, chimeric mice carrying the homeobox box 1.1 allele have been It has been created using a modification of the gene targeting method (Zimmer, A., et al., Natu re 338:150-154 (1989)). In this variant, the vector array is A linear DNA molecule containing only the hox 1.1 allele without any Integration of vector sequences was avoided by microinjecting the cells. This DN It was found that A causes gene conversion of the five alleles of cells. “Transformed "Selection to facilitate recovery of ES cells was not used; this It was identified using PCR chain reaction (“PCR”). “Proposes denaturation or sample contamination.” It is established. In these chimeric mice, the “converted” genes are passed on to their offspring. (Zimmer, A., et al.) -Current Com+*unications in Mo1ecula r　Biology'', 　Capecc■Weir A　M, R, ed., Co. Cold Spring Harbor Press, Cold Springs, New York (1989), pp. 53-58).

The use of gene targeting methods is depicted in Figure IA. This figure shows an extract of the hprt gene sequence. A gene in which the nptII gene is inserted into the son (indicated as region “3”) A construct is created. Next, this construct undergoes recombination with the cell's hprt gene. make it worse Such recombination results in disruption of the above construct to the exon 3 sequence of the cell. resulting in a replacement with the exon 3-nptII sequence. Significantly, Figure IA As shown in This results in the production of significant modifications in the sequence of the gene. As shown in Figure IA, this The efficiency of the gene targeting method is approximately 1/300.

■ Creation of chimeric and transformed animals: use of insertion vectors In contrast to the methods described above. In contrast, the present invention provides the ability to create minute, precise, predetermined irregularities in the desired genetic sequence of a cell. Can create natural mutations. The present invention has several aspects, but the most A simple one is shown in Figure IB.

As shown in Figure IB, the nucleotide sequence of the hprt gene was prepared using an insertion vector. induce mutations in. Combining this vector type with a second selectable return event nl) chromosomal disruption caused by the tII gene or vector sequence. Prevented. Therefore, the present invention avoids significant distortion of the recipient chromosome. .

Furthermore, gene targeting efficiency was significantly improved (i.e. 1/300 versus 1/3 2).

The DNA molecule to be introduced into the recipient cell contains regions homologous to regions of the cell's genome. It is preferable to have. In a preferred embodiment, the DNA molecule comprises the genome of a pluripotent cell. It will contain two regions of homology (both chromosomal and episomal). these homologous region adjacent to the “desired gene sequence” that is desired to be integrated into the cell genome. It would be preferable to do so. As mentioned above, these homologous regions are less than 2 bases long. It can be of any size as long as it is large. Most preferably, these homologous regions are 1 It will be greater than 0 bases in length.

The DNA molecule may be single-stranded, but is preferably double-stranded. The DN A molecule can be converted into DNA by reverse transcriptase or other means. It can be introduced into cells as an NA molecule. Preferably, the DNA molecules are two It would be a chain linear molecule. As the best method for carrying out the invention, a closed covalent bond is Such molecules are obtained by cleaving a fused ring molecule into a linear molecule.

Preferably, the molecule is cleaved at a single site to produce blunt or overhanging straight ends. Using restriction endonucleases that can give molecules. most preferably , the nucleotides on each side of this restriction site are linked to the introduced DNA molecule and the cellular genome. The two preferred regions of homology between the two systems would constitute a small portion.

In this way, the present invention provides a method for injecting "desired gene sequences into specific chromosomal locations in the genomes of animals and plants." ``The desired gene sequence'' can be of any length. It may have any nucleotide sequence. Also this is a complete tamper one or more gene sequences encoding a protein, fragments of such gene sequences, regulatory sequences; It may also consist of columns. The desired gene and the recipient cell's native gene are slightly different. (e.g. one base or multiple bases compared to the natural gene) Importantly, it may contain modifications, insertions or deletions. child The use of desired gene sequences such as enable you to create. Therefore, the present invention provides methods for manipulating gene expression and regulation. Provides a means for modulating.

Specifically, the present invention provides for replacing a gene sequence with a "non-selectable" desired gene sequence 1. A means of manipulating and changing gene expression and protein structure by provide. The presence or expression of a certain gene sequence in the recipient cell determines the If the gene does not provide an advantage for the survival of that cell under nutrient conditions, Arrays are said to be unselectable. Therefore, by definition, “unselectable” It is not possible to select which cells receive the gene sequence. In contrast, “dominant” inheritance A child sequence is a sequence that, under certain circumstances, can provide factors advantageous to the survival of the recipient cell. It is possible. Neomycin resistance conferred by the nptII gene is Factors favorable for survival of cells cultured in the presence of omycin or G418 It is.

Specifically, the present invention relates to genes that are present with "similar" sequences in recipient cells. Allows array substitution. two sequences are essentially similar with respect to their sequence However, there is a minute change in their mutual sequences that corresponds to a single base substitution, deletion, or insertion. or if the two sequences carry “minor” multiple base alterations, these One sequence is said to be a similar sequence to the other. of the dominant selectable marker gene. Insertions are excluded from such modifications.

The desired gene, which is flanked by regions homologous to the recipient cell, is inserted at both ends. When introduced as a linear double-stranded molecule corresponding to the homologous region of One recombination event will occur in approximately 5% of transfected cells.

A single such recombination event results in the integration of the entire linear molecule into the genome of the recipient cell. will guide you.

The structure generated by the incorporation of the linear molecule is generated by a subsequent second recombination event (one approximately 10-5 to 10-') per cell generation. This second recombination event is the integrated structure of all DNA excluding adjacent homologous regions and the desired DNA sequence? This will result in the removal of

The result of the second recombination event is therefore between adjacent homologous regions in the genome of that cell. The instability of gene replacement is eliminated by replacing the DNA sequence normally present in the gene with the desired DNA sequence The goal is to remove the

any method that would cause the introduced molecule to undergo recombination in its homologous regions. Therefore, introducing a DNA molecule containing the desired gene sequence into a pluripotent cell Can be done. Some methods include direct microinjection and calcium phosphate transformation. , which can cause the introduced molecules to form concatemers upon incorporation. these The concatemers of can disassemble themselves to form non-concatemer incorporation structures.

Since the presence of concatemers is undesirable, methods that allow concatemer formation are preferred. It's not right. In a preferred embodiment, the DNA is introduced by electroporation. (Toneguzzo, F. et al., Nucleic Ac1ds Res , 16:5515-5532 (1988); Quil et al.

J. Immunol, 141:17-20 (1988); , P, et al., Proc, Natl, Acad, Sc weir A (U, S, A,) 85:8027-8031 (1988); all of these documents (which constitutes a part of this specification).

After allowing the introduction of the DNA molecule, the cells are subjected to conventional conditions known in the art. Cultivate with

To facilitate recovery of cells that have received DNA molecules containing the desired gene sequence DNA containing the desired gene sequence and a detectable marker gene sequence. It is preferable to introduce it in combination with a second gene sequence having the same gene sequence. Purpose of the invention The presence of that gene sequence in a cell is important for the cell's cognition and clonality. Any gene sequence that allows isolation can be used as a detectable gene sequence. can be used.

In one embodiment, the presence of a detectable marker sequence in the recipient cell hybridizes , detection of radiolabeled nucleotides, or generation of detectable marker sequences. recognition by other detection assays that do not require Preferably using PCR Detecting lever-like sequences (Mullis, K. et al., Cold Spri ng Harbor Symp, Quant, Biol, 51:263- 273 (1986); Erlich H et al., EP50424; EP8 4796. EP258017. @EP237362 ; Mu Mullis, K., EP201184; Mullis K., et al., US 4683202; Erlich, H,, US4582V88: 5ai ki, R, et al.

US461113194; these documents form part of this specification).

PCR uses two oligonucleotides that are complementary to the region(s) of the sequence to be amplified. Amplification of specific nucleic acid sequences is achieved by using tidebrimers. .

Next, the extension product incorporating the above primer becomes the template for the subsequent replication steps. .

PCR is used to determine the concentration of a nucleic acid molecule, even if the molecule has never been purified before. selectively expands even when only one copy is present in a particular sample provide a method for This method can be used both for the propagation of -end-strand DNA and for the propagation of double-strand DNA. It can also be used for propagation.

Most preferably, however, the detectable marker gene sequence is and will result in a selectable phenotype. Such a preferable detectable Examples of possible gene sequences include the hprt gene (Littlefield, J. 1. , 5science 145 Near 09-710 (1964); This document (Constituting a Part of this Specification), Xanthine-Guanine Oratory ual'''', 2nd edition, Cold Spring Harbor Laboratory Press, New Iris et al., Mutat, Res. 214:223-232 (1989); (Documents are incorporated herein by reference), nptII gene (Thomas, K. R, et al. Ce1l 51:503-512 (1987); Manso ur, S, LA et al., Natur e 336:348-352 (1988): Both of these documents include a part of this specification. ), or amino acid analogs, nucleoside analogs or antibiotics, etc. Other genes that confer resistance to are included. Examples of such genes include , dihydrofolate reductase (DHFR) enzyme, adenosine deaminase (AD A), asparagine synthetase (AS), hygromarin B phosphotrans ferase or CAD enzyme (carbamyl phosphate synthetase, albaragin) encodes enzymes such as acid transcarbamylase and dihydroorotase) Gene sequences are included (Sambrook et al., “Molecular Clo ning A Laboratory 1lanual-, 2nd edition, Coldos Pring Harbor Laboratory Press, New York (191119); (This document is incorporated herein by reference).

Active thymidine kinase (TK) enzyme, hypoxanthine-phosphoribosyltrans spherase (HPRT) enzyme, xanthine-guanine phosphoribosyltrans ferase (XGPRT) enzyme or adenosine phosphoribosyl transfer Cells that do not contain the enzyme (APRT) contain hypoxanthine, aminopterin, and/or mycophenolic acid (preferably adenine, xanthine and/or or thymidine), and cannot grow in media containing thymidine. , 5-bromodeoxyuridine, 6-thioguanine, 8-azapurine, etc. can be grown in media containing leoside analogs (Littlefie ld, J, W,, 5science 145 Near 09-710 (1964 ) ; Sambrook et al., “Mo1ecular Cloning AL laboratory Manual” A 2nd edition, Ko Old Spring Harbor Laboratory Press, New York (1989)) .

Conversely, cells containing such active enzymes are capable of growing in the above media. Kiruga, 5-bromodeoxyuridine, 6-thioguanine, 8-azapurine, etc. cannot grow in media containing nucleoside analogs of Samb. rook et al., “Molecular Cloning A Laboratory y　annual'', 2nd edition, Cold Spring Harbor Laboratory Press S., New York (1989)).

Cells expressing active thymidine kinase grow in medium containing HATG but in a medium containing nucleoside analogs such as 5-azacytidine. (Giphart-Gassler, M. et al., 1 Res. 214:223-232 (1989)). active H3V -Cells containing the tk gene are treated with gangcylovir or cannot grow in the presence of similar drugs.

A detectable marker gene is a genetic marker that can compensate for a recognizable cellular defect. Anything is fine as long as it's a child. Thus, for example, using cells lacking HPRT activity In this case, use the HPRT gene as a detectable marker gene sequence. I could do that. Therefore, this gene has its expression product selected for mutant cells. or an example of a gene that can be used for “negative selection” of cells expressing this gene product. Ties, O. et al., Nature 317:230-234 (1985) ; these documents are incorporated herein by reference) are the most preferred detectable markers. This is the car gene sequence. 1, one of the genes or the hprt gene and the nptII gene. Particularly preferred are constructs containing the genes together.

A. A single DNA segment containing both a detectable marker sequence and the desired gene sequence. child usage In a first preferred embodiment, the homologous region(s) are adjacent detectable marker genes. Place the gene sequence on the same DNA molecule that contains the desired gene sequence. Provide to recipient cells. As discussed above, this DNA molecule is a linear molecule It is preferable.

Cells that have integrated the desired DNA molecule (e.g., their detectable marker gene sequence) If the nptH gene sequence is expressible, G418-resistant cells can be selected. After selection (by Check as follows. Approximately 107 cells were cultured and this was combined with the native sequence (i.e. into the recipient cells) a second recombination event (described above) resulting in the replacement of the normally naturally occurring gene sequence) Screen for cells that have caused this.

Any of a variety of methods can be used to identify cells that have undergone a second recombination event. You can stay there. Direct screening of clones, use of PCR, hybrids The use of shaping probes, etc. can all be used for this purpose. As a preferred embodiment, The DNA molecule contains the desired gene sequence, adjacent homologous regions and a detectable marker. In addition to the gene sequence, it is possible to select or recognize cells that have undergone a second recombination event. It will contain additional gene sequences to make it. This additional gene sequence It will be excised from the cell's genome as a direct result of a recombination event. did Therefore, gene sequences suitable for this purpose include those that have been deleted from cells. Any gene sequence that can be detected or selected for is included. Such “negative” Examples of "selection" gene sequences include the transgenic L1 gene and the IK gene (particularly the herpes simplex virus). In a first preferred embodiment, the frequency of the second recombination event is on the order of 10-S. death However, the use of "negative selection" gene sequences has reduced the number of such recombinant cells to approximately 10 Can be identified with a frequency of 0%.

As shown in Figure 2, the DNA molecule contains a region of heterologous structure located at the intended insertion site. may have. Insertion of such vectors allows the insertion (rather than other potential sites) It becomes possible to select recombinants that have recombined at the entry site. Insertion where recombination is desired site, thereby locating the foreign species at the intended insertion site of the DNA molecule. Deletion of the sequence of the structure (eg HSVtk in Figure 2A) will be induced. other group Insertions resulting from recombination events will retain this heterologous structural sequence. therefore, A heterologous structural region or “negatively selectable” marker that encodes an assayable gene product. By using a heterogeneous structure region that can be used as a marker, Easily determine that the locus of cellular insertion contains the exact sequence desired Can be done. As shown in Figure 2A, the efficiency of such vectors is approximately 1/197. Ru.

The heterologous structural region that can be introduced into the insertion site of the DNA molecule may be short (e.g. 26 salts). Even if the base pair is 2 (Fig. 2B) or quite large, for example 2 kb (Fig. 2A) good. The site of linearization is the 5° side, 3° side of the heterogeneous structure region, or the heterogeneous structure region. It can be within the area. Efficiency of gene targeting when the site of linearization is within a heterologous structural region is 1/63.

As shown in Figure 2C, the heterologous structural region is positioned within the homologous region where the desired recombination occurs. You may place Inheritance of cellular genes at sites other than the locus of the desired recombination site Use such constructs when you want to introduce subtle mutations into the child locus. Can be done.

B. Detectable marker sequences and desired gene sequences using different DNA molecules How to provide In a second preferred embodiment, the homologous region(s) are adjacent detectable marker genes. The gene sequence is multiplied onto a DNA molecule different from the DNA molecule containing the desired gene sequence. delivered to the recipient cell. Preferably, these molecules are linear molecules.

If provided on a separate DNA molecule, a detectable marker gene sequence and desired gene sequences by co-electroporation or other equivalent techniques. Most preferably, it will be provided to the recipient cell.

A detectable marker expressing such a receptor (preferably the nptII gene) - by conferring cellular resistance to the antibiotic G418 using the sequence After selection, grow those cells and confirm insertion events (preferably (using PCR).

All (if no selection, only 1 out of 107 cells will contain the expected recombinant construct) It can be predicted that there will be no more than a structure. However, a detectable marker sequence (dan p If recipient cells containing and expressing the tII gene (e.g., tII gene) are selected, both DN Cells absorbing A molecules can be concentrated 103 to 105 times. typical Typically, such enrichment allows screening as few as 800-1500 cells. (The introduced DNA, A, has been integrated into the genome of that cell) This makes it possible to identify recipient cells. Such screening can be done by PCR or or other equivalent methods. Using the above negative selection technique The vector copy number can be manipulated by

The two introduced DNA molecules are generally not assembled at the same site in the recipient cell's genome. Not included. Therefore, in some cases, the desired gene sequence may have been integrated in such a manner as to replace the natural cellular genetic sequence between the regions. . The locus into which the detectable marker gene is integrated is are of no significance for the purposes of this invention unless they are genetically linked to the same locus as do not have. However, if desired, DNA containing a detectable marker sequence and It is also possible to incorporate gene sequences that allow negative selection. Therefore, the introduction to finally select cells in which the selected selectable marker gene sequence DNA has been deleted. Can be done.

Preferred embodiments of the method for identifying homologous recombination events using C3 direct selection include: In both cases, one of the cell's alleles is mutated to contain the desired gene sequence. Although each embodiment allows for the isolation of desired recombinant cells, Requires screening of a significant number of candidate cells to determine. But above directly selecting the desired recombinant cells by using a variation of the embodiments described above; Can be done. This aspect of the invention is depicted in FIG. In the method illustrated in Figure 13, the star If the sequence located under the mark (*) is the neo gene, select the excision event. If we apply 6-thioguanine and 6418 selection to will be selected.

This method for direct selection of desired cells allows for the phenotypic differentiation of target and non-target cells. relies on differences and the use of a single gene that can be used for both positive and negative selection .

Typically, in any homologous recombination experiment performed with an insertion vector, , three cell populations will be generated. The first group of cells receives the desired DNA molecule It is probably a cell that has failed. This group is the basis of candidate cells that will be isolated upon completion of the experiment. will include everything. The second group of cells contains the desired gene at a random insertion site. (i.e., at the extragenic site where the mutation is to be induced) Probably. Approximately 1 out of 103-104 total cells will belong to this group.

In the third cell group, the desired gene sequence is inserted into the desired gene by homologous recombination. It is probably a cell that has integrated into the site. Approximately 1 cell out of 105-106 total cells It would belong to this group.

In the embodiment described above, the first group of cells (cells that were not transfected) are positively selected. To identify the desired recombinant cells, which can be removed by selection, Only about 1000 cells remain to be screened. In this mode is between the cells of the third group (homologous recombinants) and the cells of the second group (random inserts). If phenotypic differences exist, the cells of the third group can be selected from the cells of the second group. Wear.

Random integration sites (depending on DNA concentration when transfecting cells) likely to be concatameric with a small number of single-copy clones Therefore, such an incorporation event is inherently unstable. Therefore, something like this Concatemeric constructs will typically undergo intrachromosomal recombination. like this Recombination will always leave one intact copy of the vector in its genome. . Therefore, if a negative selectable marker is included on the vector. can negatively select all random insertion events from the population.

In contrast, the desired gene sequence is integrated into the desired gene by homologous recombination. Cells with a relatively high frequency of cell division (depending on the size of the overlapping structures) Mutations that revert at approximately 1 in 104 to 1) and do not contain vector sequences will produce the desired gene. Therefore, the vector is capable of negative selection. Even if they contain functional gene sequences, such cells will not survive negative selection. There will be some leftovers that will be able to be recovered. Most homologous recombinant cells revert will not occur and will be removed by negative selection. In this way, these selections will result in the isolation of the desired recombinant.

This method uses “precursor cells” (i.e., “desired” recombinant cells by applying this method). cells to be transformed into cells) under non-selective culture conditions or under the first selective culture conditions. It consists of incubating. Certain culture conditions (i.e., medium, temperature, etc.) progenitor cells, desired cells and intermediate cell types (i.e., precursor cells progress to desired cells) It is possible to promote the growth (or maintain the viability) of cells obtained in the process of If possible, the culture conditions are said to be "non-selective." Certain culture conditions are specific cells (i.e., have a specific genotype and therefore also produce a specific gene product in the active form) promotes the growth (or maintains the viability of cells contained in an inactive form). Cultivation conditions are said to be "selective" if the culture conditions can be maintained.

Therefore, preferred selective culture conditions depend on the genotype of the progenitor cells. mentioned above As in the active thymidine kinase (TK) enzyme, hyboxanthin-phosphoribo siltransferase (HPRT) enzyme, xanthine-guanine phosphoribosi XGPRT enzyme or adenosine phosphoribosyltransferase (XGPRT) enzyme or adenosine phosphoribosyltransferase Cells that do not contain the enzyme spherase (APRT) contain hyboxanthin, amino vitellin, and/or mycophenolic acid (preferably adenine, xanthine) and/or thymidine) and thymidine-containing media. Not possible, but 5-bromodeoxyuridine, 6-thioguanine, 8-azapurine can be grown in media containing nucleoside analogs such as. On the contrary, that Cells containing active enzymes such as 5- Nucleosylated substances such as bromodeoxyuridine, 6-thioguanine, and 8-azapurine It cannot grow in media containing the analogues.

Such incubation is carried out with D containing the desired non-selectable gene sequence. Performed in the presence of NA molecules. Preferably, the DNA molecule contains this desired gene sequence. the planned gene sequence of the progenitor cell's genome adjacent to and to the desired gene sequence Contains two additional regions of homology sufficient to undergo homologous recombination with The D The NA molecule is a selectable gene sequence that allows cells to be cultured under first selective culture conditions. Its presence or expression within the cell can be selected by feeding the and culturing the cells under the second selective culture conditions. Added selectable gene sequences that can negatively select for the presence or expression of and contain it.

Examples of preferred selectable gene sequences include active thymidine kinase (TK) enzymes. , hyboxanthin-phosphoribosyltransferase (HPRT) enzyme, xa tingin-guanine phosphoribosyltransferase (XGPRT) enzyme or Encodes the adenosine phosphoribosyltransferase (APRT) enzyme Contains gene sequences that Such gene sequences are subject to both positive and negative selection. It can be used for.

Additional gene sequences that can be used as selectable gene sequences include dihydrofolate reductase (DHFR) enzyme, adenosine deaminase (ADA), asparagus Ginncetase (AS), hygromycin B phosphotransferase, or CAD enzymes (carbamyl phosphate synthetase, aspartate transcal Contains those encoding enzymes such as vermylase and dihydroorotase) Ru.

A method for creating cells deficient in the expression of these enzymes is described by Sambrook et al. o1ecular Cloning A Laboratory Manual , 2nd edition, Cold Spring Harbor Laboratory Press, New York ( 1989); this document is incorporated herein by reference. . These gene sequences can only be used for positive selection.

Incubating under conditions sufficient to allow introduction of the DNA molecule into precursor cells. tion. The DNA molecule thus introduced is then integrated into the genome of the progenitor cell. can undergo homologous recombination with the predetermined gene sequence, thereby A desired cell in which the desired non-selectable gene sequence has been inserted into the intended gene sequence. is generated.

The cells are first cultured under selective culture conditions, and then the cells are cultured under non-selective culture conditions. cells to undergo intrachromosomal recombination and then culture the cells under second selective culture conditions. Harvesting the desired cells as described above by incubating the cells Can be done.

Therefore, in a preferred embodiment, the progenitor cells are active hypoxanthine-phosphors. holibosyltransferase (HPRT) enzyme, xanthine-guanine phospho ribosyltransferase (XGPRT) enzyme, or adenosine phosphoribo siltransferase (APRT) enzyme and the selectable gene sequence. The cells express active HPRT, XGPRT, or APRT enzymes. 1st choice Possible culture conditions include hypoxanthine, aminopterin and/or mycopherin. Containing nolic acid (preferably adenine, xanthine and/or thymidine) Use a suitable medium. In the second selectable culture condition, 5-bromodeoxyuridine Culture media containing nucleoside analogues such as N, 6-thioguanine, and 8-azapurine. Use the ground.

In a second preferred embodiment, said progenitor cell lacks an active TK enzyme and said selected A possible gene sequence expresses an active TK enzyme. In the first selectable culture condition A medium containing , hypoxanthine, aminopterin and thymidine is used.

In the second selectable culture condition, gangcyclovir (gangcyclovir) ) or FIAU (Borrelli, Proc, Nat' 1. Ac ad, Sci, (U, S, A,) 85 Near 572 (1988j) Thymidine such as (if using the H3Vtk gene) or use a medium containing 5-bromodeoxyuridine etc. (cell tk gene ).

A preferred negative selectable marker is the hprt gene (active HPRT enzyme In the presence of certain nucleoside analogs such as 6-thioguanine, cells expressing cannot grow). The efficiency of 6-thioguanine selection is cell density dependent. Therefore, when using 6-thioguanine as a negative selection agent, 104 cells/c Preferably, a density of I112 is used. Transfected cells 107 In a typical experiment using individuals, after successive selections approximately 10 revertant cells You will get it. By using “poly A selection” for the first selection, you can return The relative yield of body clones can be substantially increased. Regarding “Poly A selection” This will be discussed in detail in Example 6 below.

■ Creation of chimeric animals and transformed animals The chimeric animals or transformed animals of the present invention the one or more DNA molecules into precursor pluripotent cells (most preferably ES cells) or is created by introducing it into the isohard material (Robertson, E. , J, , “Current Communications in Mo 1ecular Bi shield attack shield ■凵h, Ca pecchi, M. R., ed., Cold Spring Harbor Press, New York. Cold Spring Harbor, Ark. (1989), pp. 39-44; (which forms part of this specification). The term "precursor" refers to the pluripotent cells of the present invention. to the desired (“transfected”) pluripotent cells created according to the teachings. It shall only be meant as a precursor. Pluripotency (precursor or transfection) transfected cells) using methods known in the art (Evans, M. J., et al. , Nature 292:154-156 (1981)). Mela animals or transgenic animals can be formed.

Any ES cell can be used according to the invention. However, ES cells Preference is given to using primary isolates. Such isolates are described by Robertso n, E, J,.

-Current Co+communications in Mo1ecula r Biology’, Capecchi, M, R, ed. A Colds Spring Harbor Press, Cold Spring Harbor, New York (198 9), pp. 39-44) directly from the embryo, such as the CCE cell line disclosed in Alternatively, clonal isolation of ES cells derived from CCE cell line C3chwartzber G, P, A, et al. 5science 2462799-803 (1989) (This document forms part of this specification). like this Loan isolation can be accomplished according to the method of E. J. Robertson. (Go eratocarcinomas and Embryonic Stem Cells: A Practical Approach'', EA J. Roberts on ed. J Press, Ofsford, 1987, this document and method are incorporated herein by reference. ). The purpose of such lawn propagation is to increase the efficiency of differentiation into animals. The aim is to obtain ES cells with higher yield. Clonally selected ES cells are transformed It is approximately 10 times more effective than its progenitor cell line CCE in producing moults. claw This selection provides no benefit to the recombinant method of the invention. clonally derived from embryos Examples of ES cell lines used are the ES cell lines ABI (hprt) or AB2.1 (hpr t-).

ES cells were prepared by E, J, Robertson (Goeratocarcinoma s and Enbryonfc Stem Ce1ls:`P Ractical Approach”, edited by E. J. Robertson. , IRL Bulletin, Ofsford, 1987.71-1P2 page; this document is incorporated herein by reference). STo cells (particularly 5NC4STO cells) and/or primary embryonic fibroblasts, etc. ) is preferred. Stolum (and/or fibroblast) cells have abnormal E Functions to eliminate clonal overgrowth of S cells. most preferably leukemia Culturing these cells in the presence of globulin factor (“Iif”) (Gough, N, M, et al., Reprod, Fertil, Dev, 1:281- 288 (1989); YaIllamori, Y. et al., 5science 246:1412-141U (1989); these (Both documents are incorporated herein by reference). The gene encoding Iif has already been cloned. Since it has been loaned (Gough, N, M, et al., Reprod, F Ertil, Dev, 1:281-288 (1989j), Transforming stoma cells with this gene using means known in the art and then Culturing ES cells on transformed stomal cells that secrete 1if into the culture medium is particularly preferred.

Logomorpha (i.e. mice, rats, hamsters, etc.), rabbits, sheep, goats, fish ES derived or isolated from mammals such as pigs, cows, primates and humans. Although cells are preferred, ES cell lines can be derived from any species (e.g. chicken). can also be induced or isolated.

■ Creation of chimeric plants and transformed plants The chimeric plants and transformed plants of the present invention A plant cell that has received a DNA molecule by use of the methods disclosed herein created through the reproduction of

Protoplasts can be isolated and cultured to achieve complete regeneration. All plants that are capable of giving plant matter contain a complete plant containing the introduced gene sequence. Whole plants (which can be transformed according to the present invention) should be harvested. The genus includes, for example, species of the following genera: Strawberry, Lotus , Alfalfa genus, Onobrychis genus, Rhododendron genus, Trigonella*, cowpea, citrus, linium (Li) num) genus, Manicot genus, Manicot genus, Carrot genus, Arabidopsis, Brassica, Radish, Nanna Sinapis spp., Atropa spp., Capsicum spp. Asus genus, Hyos genus, Tomato spp, Tobacco spp, Solanum spp, Tsukubane spp. Genus, Foxglove, Majorana, Chicorylum ( genus Cichorium, genus Helianthus, genus Cichorium, genus Cichorium, Asparagus, Snapdragon, Forget-me-not, African Orchid, Tenji genus Quaifolia, genus Millet, genus Chikarasiba, genus Ranunculum, genus Prunus genus, Salumana genus. Genus, Cucumber, Brovallia, Soybean, Lolium ( genus Lolium, genus Triticum, genus Triticum, genus Morocon, genus Sweetpotato, genus Triticum Diatomina, Cyclamen, Apple, Prunus, Rose, Rubus, Jakoya Allium genus, Santarum genus, Allium genus, Lily genus, Narcissus genus, Solanum, Bean, Bean, Trifolium, and Datura Genus Gao.

The main grain species, sugarcane, various species of the genus Euphorbia (such as beets and sugar beets), cotton, fruit including (but not limited to) trees and other trees, legumes and vegetables; ), virtually all plants can be regenerated from cultured cells or tissues. There is a growing body of evidence that it can be done.

For plant regeneration from cultured protoplasts, see Evans et al., “Protoplasts Isolation and culture of Protoplast (original title: Protoplast l5olation and Culture)”, &ndbook of Plant C■P I Curture 1:124-176 (Macmillan Publishing Company) -, New York, 1983), M., R., Davey, “Plant Protoplasts” Recent Developments in the Cultivation and Regeneration of Stroke ents in the Culture and Regeneration of Plant Protopl≠Karakuto j”, Pr otoplasts, 1983-Lecture Proceedings, Pages 19-29 (Birkhauser, Basel, 1983) A P., J. Dale, “Protoplus for Cereals and Other Difficult Crops” Protoplast Culture and Plant Regeneration (Original title: Protoplast Culture and P lant Regeneration of Cereals and 0■ ■Chichi@R ecalcitrant Crops)”, Protoplasts 198 3-Lecture Proceedings, page 31-4 (Birk Hauser, Basel, 1983), H. Binding, - Plant Regeneration (Original Title: Regeneration of Plants)-, Plant Pro toplasts, pp. 21-37 (CRC Press, Boca Seton, 1985 ) is described.

Regeneration varies between plant species, but is generally a form containing the introduced gene sequence. A suspension of transformed protoplasts is formed. Next, this protoplast Induce embryogenesis from suspension to ripening and germination stages similar to natural embryos be able to. Culture media generally contain various amino acids as well as auxins and cytokines. It may contain hormones such as Add glutamic acid and proline to the medium It is also advantageous (particularly for species such as corn and alfalfa) to be. Usually shoots and roots develop at the same time. Efficient regeneration depends on media, genotype and It will depend on its culture history. By controlling these three variables, playback Fully reproducible and repeatable.

Mature plants grown from transformed plant cells to create inbred plants Let things self-pollinate. Inbred plants produce seeds containing the introduced gene sequence do. Express this desired gene sequence by growing these seeds Can create plants.

Parts such as flowers, seeds, leaves, branches, and fruits obtained from regenerated plants can be processed according to the present invention. Included. Progeny and varieties and mutants of regenerated plants are also within the scope of the invention. Included.

As used herein, variety refers to a heritable change that is sexually transmitted to progeny plants. A stable, heritable phenotypic change that includes

■, Gene expression In one embodiment, the DNA molecule to be introduced into the recipient cell according to the method of the invention is , a plasmid or viral vector that can replicate autonomously within a host cell - (or derivatives thereof).

Preferred prokaryotic vectors include plasmids that can replicate in E. coli (e.g. For example, pBR322, CoIEl, pscol, pAcYc181, πvX, etc. ) and other plasmids. Such plasmids are for example Maniati s, T, et al. (1o1ecular Cloning, ^Laboratory 1lanual', Cold Spring/%-/<- Breath.

Cold Spring Harbor, New York (1982)) There is.

Bacillus plasmids include pc194, pc221, pT127, and the like.

Such plasmids are described by Gryczan, T. "ology of the Bacilli", Academic Press, New York (1982), pp. 307-329). Preferred spot The Treptomyces plasmid contains pIJ101 (Kendall, K. , J, et al., J, Bacteriol, 169+4177-4183 (198 7)) and φC31 (Chater, K, F, et al. -3ixth In terna Koenshi Shield Lion ■ I Symposium on Actinomycetales Biolo gy”, Akademiai φ Kaito - Budapest Hungary (1986), 45- Streptomyces and cteriophages such as p. 54) are included. '/s- tomonas plasmid by John, J., F., et al. (Rev, Infe ct, Dis, 8:693-704 (1986)) and Izaki, K. , (Jpn, J, Bacteriol, 33 Near 29-29-742 ( A review of 1X7 be.

Suitable yeast vectors include the yeast 2 micron circular, expression plasmids YEP13, Y Included are CP and YRP or derivatives thereof. Such plasmids are Well known in the technical field (Botstein, D. et al. tr, Sy+sp, 19+265-274 (19g2): Broac h, J, R,, he Mo1ecular Biology of th e Yeast Sacchar Shield Takakamo ■ Katana @: Lif e Cycle and Inheritance-,)-Led Spring/ \-/<-Laboratory, 2x-Cold Spring Harbor, York, 44 pp. 5-470 (1981); Broach, J. R., Ce1l 28 2203-204 (1982)).

Examples of vectors that can be used to replicate DNA molecules in mammalian hosts include the bovine papillomavirus, polyomavirus, adenovirus or SV40 virus Contains animal viruses such as Rus.

■1 Use of the present invention The method of the present invention allows the introduction of a desired gene sequence into animal or plant cells. enable.

In a first embodiment, a chimeric animal or transformed cell containing the desired gene sequence The method of the present invention can be used to introduce DNA into the germline cells of an animal to create a Can be used. can be created by applying the method described here Animals include chickens, non-human mammals (particularly members of the Order Rodentia (i.e. mice, rats). , hamsters, rabbits, sheep, goats, fish, cows, non-human primates). included.

As mentioned above, the desired gene sequence can be of any length and of any length. It may have a nucleotide sequence such as Specifically, a preselected cell In order to create single or multiple base alterations, insertions or deletions in a gene, A desired gene sequence can be designed.

If such changes are within the translated region of the natural gene sequence, the natural protein New protein variants of proteins can be obtained. For example, improved (i.e. , more stable, more active, etc.) enzymes, binding proteins, receptors, receptors Using such techniques to create animals that produce ter-ligands etc. Can be done.

The method of the present invention is used to create cells in which natural genes are replaced with foreign structural genes. The law can be used. A given gene is from a different species than that of the transformed cell. If it is inducible, the gene is considered to be a foreign construct to the transformed cell. It will be done.

In one embodiment, this substitution can be accomplished in one step crg:I3).

To accomplish such a replacement, one that contains the desired gene sequence and homologous regions must be A DNA molecule is introduced into a recipient cell. a selectable marker gene in the cell. This is also used to select cells that have undergone recombination. This method is , a heterologous structural sequence having the desired DNA sequence of the normal sequence adjacent to the homologous region; results in the replacement of

In a second embodiment, this substitution can be performed in two steps (Fig. 4). Aspects mentioned above Similarly, providing cells with a DNA molecule containing the desired gene sequence and homologous regions do. When a recombination event occurs, this DNA molecule is introduced into a homologous site. a selectable marker used to select cells that have been inserted into the chromosome in It also contains genes. The structure of such an insertion site is shown in FIG. 4A.

Importantly, in this embodiment, the introduced DNA molecule contains a "negatively selectable" marker. - will also contain genes. This “negatively selectable” marker gene Select cells in which the recombination event of step 2 has occurred resulting in the deletion of the inserted DNA. can be used for.

As shown in Figure 4B, using a second DNA molecule to complete the gene replacement Ru. This second DNA molecule must contain some selectable marker gene. There isn't. Upon receipt of the second DNA molecule, a second recombination event occurs and the “second a “first” DNA molecule that is integrated with the “first” DNA molecule (this is the desired DNA sequence) sequence, selectable marker sequence and “negatively selectable” contained in the molecule. (including marker sequences) are exchanged. This aspect of the invention is illustrated in Figure 4B.

Another aspect of the invention includes a positive selection marker (nptII gene or and a negative selection marker (such as the 1st district gene). Introducing small mutations at desired loci by using “set” constructs can do. In this embodiment, the positive selection ability of the construct is first used to and two selectable markers are introduced into the desired genetic locus. Then add the desired mutation to By providing cells with DNA molecules containing (replacements, insertions, deletions, etc.). (using a negative selection marker) Contain the desired mutations in your cassette ° sequence by selecting deletions in the A recombination event can be selected that results in a replacement of the DNA sequence. present invention This aspect of is represented in FIG.

The method of the present invention can be used to replace adjacent regions of a chromosome with a desired gene sequence. You can also use the method. Therefore, the present invention includes D which may be modified or substituted. There are no restrictions regarding the size of the NA area. This aspect of the invention can be described in a five-step sequence. The operations are shown in FIG. 6 as (FIGS. 6A to 6E). What kind of gene sequence can this method be used for? can also be applied. This is a heterologous structural immunoglobulin (a major immunoglobulin gene). It is particularly useful in creating cells containing cells such as loci).

The first step in replacing a large region of a chromosome with the desired gene sequence is to identify the initial target. Contains settings. This step contains the "first fragment" of the entire desired replacement sequence. DNA molecules are given to recipient cells (Figure 6A). “First fragment” of this desired replacement sequence has a selectable marker sequence (most preferably the nptI engineered gene) at its end. contains.

This DNA molecule also contains genetic sequences for which both positive and negative selection methods exist. It also contains "dual selection" gene sequences encoding non-functional fragments. hprt- When used in connection with cells, an example of such a gene is the parturition ↓ gene. machine Cells that are functional and express one gene are determined by their ability to grow in HAT medium. can be selected. Cells lacking a functional cellulose gene have their 6-ti It can be selected based on its ability to grow in the presence of oguanine.

Homologous recombination results in the insertion of the DNA molecule into the cell genome at a homologous region. (Figure 6A). Importantly, this step contains a selectable marker gene It is possible to select the desired recombination event as it results in the generation of cells with the genome. It is Noh.

The second step in nature is to provide the cells with Atsushi 2 DNA molecules. This second DNA The offspring will have a functional gene product due to the internal deletion along with a “second fragment” of the desired replacement sequence. Contains the sequence of a double selection gene that cannot be encoded. Homologous recombination is a functional a double-selected gene that results in the insertion of a second DNA molecule into the genome of the cell. (Figure 6B). Recombination also allows for the integration of non-functional fragments of dual selection genes. Ras. Importantly, this step is necessary to ensure that genomes containing functional dual selection genes are It is possible to select the desired recombination event as it results in the generation of cells.

The third step in nature provides the cells with a third DNA molecule. This third DNA The offspring contains both the "first" and "second" fragments of the desired replacement sequence. The modification involves adding a third DNA to the cell genome in a manner that deletes a functional dual selection gene. results in the insertion of molecules. Non-functional fragments of double selection genes (also generated in the second step) ) is unaffected by this recombination and is retained (Fig. 6C). importantly , this step results in the generation of cells with genomes lacking the dual selection gene, so It is possible to select the desired recombination event.

The fourth step in nature provides the cells with a fourth DNA molecule. This fourth DNA The child is the “third fragment” of the desired replacement sequence and, as in the second step, a mechanism due to an internal deletion. Contains the sequence of a dual selection gene that cannot food a functional gene product. homologous Recombination adds the fourth DN to the cell genome in a manner that produces a functional dual selection gene. resulting in insertion of the A molecule (Fig. 6D). Recombination also causes non-functionalization of double-selected genes. It also results in the insertion of fragments. Importantly, this step generates functional dual selection genes. Select the desired recombination event as it will result in the generation of cells with the containing genome Is possible.

The fifth step in nature provides the cells with a fifth DNA molecule. This fifth DNA The child contains both the "second" and "third" fragments of the desired replacement sequence. homologous Recombination adds the fifth DN to the cell genome in a manner that deletes a functional dual selection gene. resulting in the insertion of the A fragment. Non-functional fragments of double selection genes (generated in the fourth step) (Fig. 60) is unaffected by this recombination and is retained (Fig. 60). important things , since this step results in the generation of cells with genomes lacking the dual selection genes, It is possible to select the desired recombination event.

As will be appreciated, the net effect of the above steps is to to contain "first", "second" and "third" "fragments" in a sequential manner. The goal is to create cells with engineered genomes. in the genome of this cell These steps can be repeated as many times as necessary to introduce additional “fragments”. Ru. In this way, heterogeneous constructs that could not be introduced using a single vector can be Cells containing synthetic genes, chromosomal fragments or chromosomes can be constructed. Up As mentioned, each stage of nature is selectable.

Specifically, "anthropomorphic" antibodies (i.e., non-human antibodies that are non-immunogenic in humans) Robinson, R, R, et al. 1 International Patent Publication P CT/U S 8610 2269; Akira, L et al., European Patent Application 184187: Tani Guchi, M., European Patent Application 171496; Morrison, S Le, et al., European Patent Application 173494: Neuberger, M, S, et al., PCT Application WOg6101533; Cabilly, S. et al., European Patent Application Patent application 125023; Better, M. et al., 5science 240 :10104l-1043(198; Liu, ^, Y, et al., oroc, N atl.

^cad, Sci, USA84: 3439-3443 (1911t7) ; Liu, A, Y, et al., J, ImmunoL 1R9:3521-3 526 (1987) ; Sun, L, K, et al., Proc, Natl, ^cad, Sci, USA 84:214-218 (1987); N15 ■ Weir {aura, Y, et al, Can c, Res, 47:999-1005 (1987): food, C, R, et al. Nature 314:446-449 (198T)): 5 ha v et al., J.

Natl, Cancer In5t, 80:I553-1559 (198g )) This aspect of the invention can be used to create.

Also, animals with better resistance to disease, or constituting an improved food source. produce animals that provide fibers, leather, etc. with more desirable characteristics. You can also use your true nature to accomplish something. Also new animal models of human genetic diseases. You can also use nature to create dels. For example, using the nature of an animal, C Anthropomorphizing the D4fi body could provide an animal model for AIDS Can be done. By using such animal models for drug testing, genetic disease can facilitate the development of new therapies.

Furthermore, the present invention provides for medically or clinically important heterologous structural genes containing mutations. It also allows the formation of cells or transformed animals with A gene is human or If it expresses an isotype of a protein that is associated with a disease or condition in animals. A gene is said to be of medical or clinical importance. An example of such a gene is topoisomesase p180, 5-α reductase, ACAT, 5-lipoxy genase, insulin receptor, interleukin-2 receptor, epidermal growth factor receptor, seratonin receptor, dopamine receptor, GABA receptor protein, Vz vasopressin receptor, G protein (signal transduction), phospholipa This includes the genes encoding the enzyme C protein and insulin. For microinjection Therefore, the transgenic mouse that expresses human insulin was created. R, F, et al. (European Patent Publication No. 247494: This document forms part of this specification. reported by

Using the above-mentioned transformed cells and animals to study the regulation of human genes. can be done. For example, topoisomerase p180.5-α reductase, ACAT, 5-lipokingenase, hormone receptor or cytokine receptor Transgenic animals expressing human isotypes of pigs can be used for in vivo drug screening would have uses in Expression of topoisomerase p180 is associated with chemotherapy related to resistance to Therefore, substances that interfere with this enzyme should be used in chemotherapy. could be used to increase effectiveness. 5-α reductase (special Animals (particularly rats) that can express human isotypes (those in the prostate) ) would be highly desirable. ACAT is a key enzyme in lipid metabolism; Animal models of regulation would be extremely valuable. 5-Lipoxygenase Expressing animals are used in many research programs, especially screening for isotype-selective inhibitors. ) can be important for Human hormone receptor protein or site Animals expressing kine receptor proteins are known to be agonists and antagonists of receptor action. It would be of value in identifying anti-drugs. Similarly, components of the human signal transduction system (i.e., G proteins and phospholipase C). It can be used to study the pathophysiological consequences of functional damage to proteins. Dew.

Transport proteins (i.e., membranes in the gastrointestinal tract, blood-brain barrier, kidneys, etc.) that transport proteins and other molecules and ions. Expressing human proteins (proteins that facilitate or allow the passage of The present invention can be used to create cells and animals that can Next, the generation cells or animals mentioned above to study the role of such proteins in metabolism. can be used. Specifically, the protein structure or protein sequence. by such isotypes to investigate the pharmacokinetic consequences of specific differences. The degree and mode of conjugation mediated can be studied. gelcolonidetran Spherase, glycine conjugation and sulfation, methylase, and glutathione Conjugates are an example of an enzyme of particular interest in this regard.

Purification of many compounds is mediated by esterases. This kind of purifying effect When studying cells expressing heterologous structural isotypes of such esterases, Or animals can be used.

For research on the process of azo or nitro reduction, Cells or animals expressing the isotype of the protein involved would be desirable .

Significantly, some potential therapeutic substances induce toxic effects in animal models; It is often found that other animal models do not induce toxic effects. child In order to elucidate the potential of substances such as It is often necessary to determine the toxicity of the intermediate metabolite. The present invention Allows for the creation of transformed cells or animals that may facilitate such determinations do.

The methods of the invention can be used to make modifications to regulatory regions of natural gene sequences. can. The present invention therefore provides for the natural gene sequences regulated by their regulatory regions. provides a means of altering the nature or control of transcription or translation of. For example, fee Introducing mutations that remove back-back inhibition and therefore result in increased gene expression. It is possible to enter Similarly, to reduce gene expression, It is also possible to impair transcriptional ability. Such modifications are used as gene therapy techniques. It is also of particular value in developing improved animal models of human disease. example The ability to increase transcription or translation of the insulin gene, for example, is a potential genetic cause of diabetes. Provide gene therapy. Similarly, impairing the synthesis of β-globin chains can also impair β-globin chain synthesis. Provides an animal model for thalassemia.

The methods of the invention, apart from their use in veterinary and human medicine, Can be used to study gene regulation, expression and organization in animals can.

Since the method of the present invention utilizes DNA repair and recombination processes, it is possible to inhibit or Damaging substances may act by influencing these processes.

Substances that impair DNA repair and recombination potentially have use as anti-neoplastic agents As such, the present invention provides a means to identify novel antineoplastic agents.

Furthermore, both cloning of gene sequences and mapping of chromosomes or chromosomal aberrations The present invention can also be used to facilitate this.

the desired gene sequence is homologous or similar to some natural gene sequence in the recipient cell The method of the invention does not require that an animal contain and express a foreign gene sequence. enables the creation of If cells express similar genes, such similar details Desired gene sequences can be expressed in addition to the cell genes (e.g., animals can express It is possible to express both the anthropomorphic “anthropomorphic” receptor and its natural analogue. can). Thus, for example, the present invention provides important human proteins, e.g. interferon, tissue plasminogen activator, hormones (insulin and Create animals that express blood factors (e.g., growth hormone) and blood factors (e.g., factor ■) provide the means.

In a second aspect, the present invention can be used to create chimeric or transformed plants. After the method is used to introduce DNA into plant cells, it can be manipulated.

Plants that can be created by applying the methods disclosed in Uni include multicellular higher (i.e., non-fungal) plants. A non-fungal plant is any plant that is not a fungus or yeast. refers to plants.

In a third aspect, to provide treatment for genetic diseases (i.e. "gene therapy"), For introducing DNA into somatic cells of animals (especially mammals including humans) or plants The method of the present invention can be used for. The principle of gene therapy is 01dhat R, K.'s book "Pr1nciples of Biotherapy", Sheba Empress, New York, 1987) and similar textbooks.

In this third aspect, disease-causing genetic lesions are encoded with preferred gene products. Replace with the gene sequence to be coded. Examples of such genetic damage are cystic fibrosis, phenylketonuria, hemophilia, 7-year-old Willplant disease, sickle cell anemia, cod Semia, galactosemia, fructose intolerance, glycogen storage disorders, high cholesterol Rollemia, juvenile diabetes, thyroid dysfunction, Alzheimer's disease, Huntington These include Bond's disease, gout, and Riesch-Nyhan syndrome (Bondy, P., L. et al. , “Disorders of Carbohydrate iletaboli sm”, pp. 221-340, Salanders (1974); Coleman ,　i,et al.

“Mo1ecular Mechanisms of Disease”, -r --Le University Press, (1975)). Gene therapy methods and uses Boggs, S, S, (Int, J, Ce1l C1on, 8:gO-96 (1990)), Karson, E., M. (Biol, Reprod, 42:39-49 (1990)), Ledley, F., D. ,, “B Amakusa Shield ■Mo■ Shidan Geshishi■凵 A＾ Comprehensive Treatise”, Volume 7B, -Gene Technology-, V CH Publisher, Inc. State of New York, pp. 339-458 (1989)). (All of these documents are incorporated herein by reference).

In a fourth embodiment, the recipient animal or plant is treated with a virus, insect or herbicide (plant). The present invention provides a treatment for protection against pesticides, toxins, etc.) method can be used. In this embodiment, the introduced gene is for example, increased amounts of enzymes or other proteins that can degrade herbicides or toxins. to provide a gene sequence capable of mediating expression or de novo expression thereof. Dew. For example, genes that can mediate increased expression of chitinase or its de novo expression. Increased resistance to insect parasites by imparting trochanteric sequences to plant cells can be granted.

When providing a desired gene sequence to animal cells, a pharmaceutically acceptable carrier ( That is, liposomes, etc.) are preferably used. Such gene sequences are known Pharmaceutically useful compositions can be prepared by formulating according to the method described in . this By formulation, these substances or their functional derivatives are pharmaceutically acceptable. It is mixed with a carrier excipient. For suitable excipients and their formulation, see e.g. N1colau, C, et al. (Crit, Rev, Ther, Drug Carrier 5yst, 6:239-271 (19W9): This document (which is incorporated herein by reference).

To prepare a pharmaceutically acceptable composition suitable for effective administration, the composition may be It will contain an effective amount of the desired genetic sequence along with appropriate amounts of carrier excipients.

Additional pharmaceutical methods can be used to control the duration of action.

Controlled release preparations contain the desired gene sequence (conjugated to a carrier or unconjugated to a carrier). by using polymers to complex or absorb can be achieved. Controlled delivery can be achieved by using macromolecules suitable for controlled release (e.g. Polyester, polyamino acid, polyvinylpyrrolidone, ethylene vinyl acetate methylcellulose, carboxymethylcellulose or protamine sulfate ) and the concentration of macromolecules as well as the mixing method. controlled release Another possible way to control the duration of action by means of polyester preparations is to ester, polyamino acid, hydrogel, polylactic acid, or ethylene/vinyl acetate The incorporation of drugs into particles of polymeric materials, such as polymers. alternatively , instead of incorporating these drugs into polymer particles, e.g. technology or interfacial polymerization (e.g. hydroxymethylcellulose or gelatin, respectively) microcapsules and poly(methyl methacrylate) microcapsules) These substances can be encapsulated in microcapsules prepared by e.g. Liposomes, albumin microspheres, microemulsions, nanoparticles and nanocarriers these substances in colloidal drug delivery systems such as capsules or in macroemulsions. can be included.

In a fifth aspect, improving the food or fiber properties of plants or non-human animals The method of the invention can be used to For example, total protein synthesis plants or non-human animals that grow faster by increasing levels resulting in the creation of plants and non-human animals of increased food value. This method can be used to

Having now generally described the invention, reference is now made to the following examples to further illustrate the invention. It will be easier to understand. The following examples are included for illustrative purposes only. They are not intended to limit the invention unless expressly stated otherwise.

Electroporation was performed as follows.

Preparation of DNA Purify DNA used for electroporation by CsC1 gradient centrifugation did. This can be achieved by incubating the DNA with appropriate restriction enzymes. A large-scale digest of purified DNA was prepared. 500ng of this large amount of digested material is made into a mini gel. Its complete digestion was tested by multiplying by DNA concentration of large amount of digested material The concentration must not exceed 1 μg/μm.

Next, this large amount of digested product was extracted once with an equal volume of phenol/chloroform, and an equal volume of The mixture was extracted once with chloroform. Precipitate the DNA with 2.4 volumes of ethanol , pelleted by centrifugation and dried using Speed-Vac.

Next, the pelleted DNA is added to the desired concentration (usually 1 μg/μm) at 0.05 μg/μm. IXT Resuspend in sterile 5-EDTA buffer such as E (1 electroporated 25 μm of DNA per section). Next, the DNA concentration was measured using a fluorometer.

Preparation of cells for electroporation E. J. Robertson method (Goeratocarcinomas and Embryonic Stem Ce1ls: ＾orac tical Approach”, edited by E. J. Robertson, IR ABI specification according to L.Bress, Ofsford, 1987.71-112). Embryonic stem cells of the vesicle lineage were cultured to approximately 80% full growth. Expressing lif in the culture medium Cells were cultured in the presence of stomal cells. 1 day before electroporation: 2 and inoculated 4 hours before harvest.

Cells were harvested by trypsinizing them and resuspending them in medium (2× Cells from 10CI11 plates were combined into a 15vl tube for a total IQml. ).

Cells were pelleted by centrifugation and the supernatant was removed by aspiration. next , the cells were resuspended in 10 ml of phosphate buffered saline and 20 μm were counted. The total number of cells was determined by. Typical yield is 30 per 10cm plate ×106 cells.

These cells are then pelleted by centrifugation, and the supernatant is removed by aspiration. Removed. Cells were resuspended at a density of 11X10' cells/ml. That 20 μm This cell density was confirmed by counting.

electroporation Cells prepared as above were incubated in a 15ml tube in the presence of an appropriate amount of DNA. Cubated. For each electroporation, 9 ml of 25 μm DNA and Q cells were used.

The mixture was incubated at room temperature for 5 minutes (this step may be omitted). ).

This cell/DNA mixture was then carefully electroporated into a tube. (1 kg; Q per pet, 9 ml; this volume is important).

The cuvette was placed in a metal electroporation holder with a thin-piece electrode. It was placed in contact with the retaining fastener.

Bio set to 230V, 500μF (this requires a capacitance extension) Electroporation was performed using a Rad Sheen Pulser. The time constant is 5 ．． It should indicate between 6 and 7.0.

Leave the cuvette at room temperature for 5 minutes, then add the cells to the appropriate density (2X 10' cells). /100mff1 plate or up to 6X10' cells/6011111 plate ) to inoculate the plates. G418 takes 3-4 days to start killing cells; G418 was used as a selective agent since the plate would have grown beyond full surface growth. Do not exceed this cell density if When to apply G418 selection It is applied 24 hours after electroporation. G418 concentration is must be titrated every time.

During the first 6-7 days (the colonies become visible and most of the cell debris has been removed) The plates were re-fed daily with fresh medium +0418 until removed). FIAU When using (0,2 μM) selection, this can proceed simultaneously.

RV 4, O (Thomas, LR, et al. Ce1l 51:503-5 12 (1987)) is 104 colonies/107 cells/100 +am plate. This yield is higher than that obtained using other constructs. Although the amount may differ significantly (and surprisingly), the use of this method always Resulting in the recovery of several colonies of cells containing borated DNA.

On the 8th, it turns red depending on where you choose the colony. Select colonies around 10-11 days It is most preferable to remove it. However, 18-21 of electroporation Colonies can be collected up to a day later.

To illustrate the invention, five Using the kb HPRT vector AD8 (linearized with Sac I) , embryonic stem ("ES") cells were co-electroporated (Figure 7). electro poration (230V, 500μF) of Q, 9ml of CCEp24 cells (7, The molar ratio was 5 x 10' cells/1) DNA: HPRT DNA). Gave The total amount of DNA was either 25.50, 100 or 200 μg. this The vectors used in the experiment are clearly shown in Figure 7. The results of this experiment are shown in Table 1.

Table-1 Simultaneous electrophoresis of gene sequence and hprt gene sequence Average number of colonies generated per 0' cell 1:10 460 3 16 0 5 g, 70 7 nd nd, nptII DNA molecule and hprt DNA It is shown that recombination of both molecules results.

The frequency of recombination is shown in Table 2 below. B 1:1 100 10.1 1.0 1/1010C1:100 200 0.8 0.2 1/400*The reaction was carried out as described above. Reproducibility of experimental results Examined. The results of this experiment are shown in Table 3.

once the sac Philippine colony G418R () transfected Neo: D NA Number of times (total number) per cell) 50 3' 192010 67 1.10 200 16 60g/7 1/868 43, 471:100 200 5 400/1 1/400 8100 7 30010 4.5 Example 3 homologous recombination Vector recombination of the above-mentioned vector into the chromosome of the recipient cell. The following experiment was conducted to investigate the structure of the body.

For this purpose, we included a 5.5 kb region homologous to the cellular hprt locus. A vector was used. This vector uses the 1ptII gene as a selectable marker. It also contains genes. Linearize this vector with ES cells were fed by troporation. G4181: Became resistant to By selecting cells and analyzing their DNA, it is possible to detect the expected integration. It was determined whether the selected structure contained restriction fragments that matched the structure.

The vector used and the expected integration structure are shown in Figure 8 (; represents the regulation of cellular DNA. G418-resistant cell power (Figure 8 (=hprt structure shown) was determined by gel electrophoresis of the limited digest. It was confirmed that it contained structure. The results of this investigation indicate that the vector can be used to stain the cells. The size of the homologous region carried by vectors in the body determines the effect on recombination frequency. Established. 5. homologous to the hprt locus. Recombinant vector containing gkb Fish were prepared as described in Example 3. Using the same method, the hprt gene A recombinant containing a similar vector with less than 1.3 kb of homology with the locus was also prepared. Manufactured.

The structure of the insertion site of this 5,8 kb vector is shown in FIG. The above two constructs Table 4 shows the return frequency. The structure obtained by returning the insert is shown in FIG.

Target frequency of insertion and replacement vectors Using a series of different vectors, this study The target frequency achieved by using the method of the invention was investigated. These vectors The tar contains 6°8 kb homologous to the murine hprt gene, and contains the linearization site or It had a region of heterogeneous structure inside (Fig. 2).

For this purpose, 108 cells were electroporated into ES cells prepared as described above. and inoculated into 10×90 plates. After 24 hours, G418 (350μ g/ml) was added to the medium. After 5 days of selection, 10-5M6-thioguanine 9 plates and 1 plate containing G418 selection as a transfection control. I left it under selection. The selection continued for another 7 days. At this point, mark the colony. The colonies were expanded as isolated clones for Southern analysis. target efficiency are detailed for each of these vectors (Figure 2 = Table 5).

Southern analysis showed that most of the 6-TG” clones were digested with CH1ndIII. It was shown to have the expected built-in structure as depicted.

Reversion of these hprt clones was performed by measuring HATlt.

Initial recombinant deletion of duplicate elements that gives rise to large numbers of colonies by cell division In order to prevent the “jackpot” effect caused by this, Cells were clonally expanded under 6-TG selection. When we obtained 107 cells, their Cells were reseeded on 90 plates without selection for 48 hours. 48 hours later, H Application of AT selection resistant colonies were recorded 10 days later and showed that typical In this case, 20-200 colonies were observed per 107 cells inoculated (Table 4). each Colonies tested returned at similar frequencies.

Table 5 Replacement vectors and insertion vectors: target and frequency genes homology vectors frequency Hox2.6 3.2kb IV+ 1/33Rv: Replacement vector, IV: Insertion Increases selection of cells that have integrated vector-transduced DNA by homologous recombination. "PolyA selection" can be used to advance.

If the introduced DNA molecule were to be randomly integrated into the host chromosome, one In general, it should not be incorporated into sites adjacent to the essential 3° polyadenylation site. Probably. Therefore, transcription of such randomly inserted constructs results in The resulting mRNA will generally lack polyadenylation. is incorporated next to the natural polyadenylation site (i.e., random insertion The use of vectors that allows for selection of recombination events (not due to homologous recombination) You can take advantage of this fact by being present.

Located in the middle of 9. One HinDIII site is present within exon 9 and one E A coRI site is located behind the end of this exon.

The first vector used contained a 5.0 kb region and therefore contained a region of exon 9. It contained a polyadenylation site (vector 6, Figure 10). As shown in Table 6, The insertion frequency was high (i.e., the frequency of G418-resistant colonies was 24×10−5). Only 1/941 colonies showed G418 resistance. However, There's some random integration going on.

Similarly, it contains the DNA from the XbaI site to the EcoR1 site of vector 6. When a 3°5kb vector (vector 10) was used, the frequency of insertion was high. (i.e., the frequency of 0418-resistant colonies was 21×10−’) The double thioguanine resistance and G418 resistance that characterize the recombinants are 1/770. Only colonies showed (Table 6). This result has some random embedding It is clear that something will happen.

However, using a vector lacking the polyadenylation site of exon 9 (i.e., vector 9), If used, random integration does not result in expression of a functional nptrz transcript. .

Therefore, the frequency of G418-resistant colonies is low (1,4 x 10-'). Landa The overall frequency of recombinant recovery as desired is reduced because the number of colonies that demonstrate undesirable integration is suppressed. degree (i.e. 1/100 overall efficiency for dual resistant colonies (Table 6)) ). The polyA selection thus results in an increase in overall efficiency of nearly 10 times. However, Colony screening to distinguish between random and homologous recombination In situations where it is desired to minimize or avoid the can be used to advantage.

Table 6 Poly A selection Vector size G418' TG" TG'10418'1 number (kb) (XIO-') (XIO-') 6 5.0 24 2.5 1/9419 3.0 1.4 1.4 1/10010 3.5 21 2.7 1/770 The method of the invention was further illustrated by using the method of the invention to .

The c-src locus contains several exons, which are labeled as “squares” in Figure 11. The regions 2 and 3 are shown as encircled areas. As shown in Figure 11A, exon 3' The natural allele of does not contain a HindIII site.

The sequence of part of exon 3' is shown in FIG. 110. As shown in Figure 110, this A 9 bp insertion into Son will result in the formation of a HinDIII site.

To accomplish this change in exon 3', one vector (src14 ) was prepared. As shown in Figure 11B, the 5rc14 vector It is homologous to a region of the constellation Genericus. However, the exon 3' sequence of this vector is H1 Modified to contain the above 9 base pair insert necessary to create the ndIII site. (Figure 110).

The second vector containing nptII (PGKneo) and the 5rC14 vector , total DNA concentration 25μgoal and molar ratio 1:5 (ne to target vector ES cells by simultaneous electroporation using the method described above at introduced into cells.

To select recombinant cells that have integrated the nptII gene, cells were 8 for 12 days. Next, we used PCR to identify the natural exon 3° Gene locus replaced with HinDIII site-containing exon 3' sequence of 5rc14 vector For cells that have undergone recombination events that result in Cells were screened.

by PCR screening using probe B and probe C (Figure 11B). Southern analysis of colonies identified in It was revealed that the protein contained the HinDIII site as desired. child Experiments demonstrate that microscopic insertions can be introduced into any cellular gene. It was done.

The ability of the present invention to introduce complex targeted mutations into the genome of recipient cells is further illustrated. As shown in 12A, the natural allele of exon 3° is either the Nhe1 site or the Ec .

Contains no RT site. However, as shown in Figure 12C, the natural If you replace the sequence ACCTGG TTC with the sequence TAG CTA GCT, N A heI site will be created. Similarly, ACAf-GAA in exon 3′ Substitution with will generate an EcoRI site (Figure 120).

To achieve these changes in the sequence of exon °', one vector Modified to contain the above substitutions (Figure 12C).

5rc33 vector in concert with a second vector containing the rltII gene. It was introduced into ES cells by the method described above. Recombinant cells containing the nptII gene To select for cells, cells were cultured in the presence of 0418. Next, using PCR Therefore, the natural exon 3°′ locus was replaced with exon 3′ of the 5rc33 vector. For cells that have undergone recombination events that result in Cells were screened.

Identified by PCR screening using probe A and probe C (Figure 120) Southern analysis of the isolated colonies revealed that the native exon 3 locus was as desired. It has been demonstrated that it has been modified to contain both an Nhe1 site and an EcoRI site. It was.

This experiment demonstrated that it is possible to introduce minute substitutions into any cellular gene. It was.

Although the invention has been described in connection with particular embodiments thereof, further modifications are possible. This application does not cover any variations, uses, or adaptations of the invention generally in accordance with its principles. These include information from this disclosure that is known or commonly used in the technical field related to the present invention. and deviations from the present disclosure as may apply to the above-mentioned essential features and including deviations from this disclosure that fall within the scope of the claims of this application) It will be understood that this is intended.

1^ Replacement vector: 6,8 kb homologous structure IB insert targeting the hprt locus. Input vector: 6.8 kb homologous structure Fl (, LINE l ^ Insertion vector with a heterologous structure at the insertion site: 2kb insert 2B at the linearization site Insertion vector with a heterologous structure at the insertion site: 26 bp insert FI at the linearization site CIJRE 2 Insertion vector with 2C added heterologous structure: homologous structure internal picuary 2 ( continuation) A 1st step, homologous recombination: addition of human substitutes by insertion vector: mouse non- code exon Mouth human non-coding exon zz human code exon Shinshi mouse code exon FIC,URE @ ncuo 4 (continued) Selective introduction of small and large genomic changes First step - standard substitution bases ctor Plan 2 Step A - Introduction of small mutations complete or partial replacement FICLJRE 5 6^ 1st stage initial target setting Fragment B, FIClJRE & Stage 3: A-B junction repair, selection (100%) A+B junction junction in 6TG Cree gpt gene F) GURE6 (continued) Step 5: Repair of B+C mating clones, selection in 5TG (100%) FICL JRE b (continued) Simultaneous double chotroporation Xh.

4bp Pvul insert F) GURE 7 Predicted structure of the hprt locus after homologous integration of the IV68 vector Cυ1'L X Reversion Fl of homologous recombinants generated by the insertion vector (, URE 9 6□　5. Okb 9 3, Okb 10□ 3.5 kb FIGURE 10 ■ abstract Contains the desired gene sequence inserted into the predetermined gene sequence by homologous recombination. A method of creating animal cells with The final method provides detailed and accurate information on gene sequence and expression. Enables the production of modified animal cells.

international search report LLIImelIIt^-1” FCT/US91104006 United States 7703 0 Texas, Hyuston, North Brace 3

Claims (37)

[Claims] 1. a desired non-selectable gene inserted within a predetermined gene sequence in the genome of a cell A method of obtaining a desired animal cell or non-fungal plant cell containing a child sequence, comprising: A. ．． The progenitor cell is a DNA molecule containing the desired non-selectable gene sequence. and the desired gene sequence is matched with the predetermined gene sequence of the genome of the progenitor cell. Two homologous regions adjacent to the desired gene sequence sufficient to undergo homologous recombination and a DNA molecule further containing B. of the DNA molecule C. The introduced DNA molecule contains the precursor particles. by causing the genome of the cell to undergo homologous recombination with the planned gene sequence. and the desired non-selectable gene sequence is inserted into the predetermined gene sequence. D. collecting the desired cells; A method consisting of things. 2. Claim 1, wherein said DNA molecule contains a detectable marker gene sequence. the method of. 3. By subjecting the precursor cells and the DNA molecules to electroporation. 2. The method of claim 1, wherein said DNA molecule is introduced into said precursor cell. 4. In step B, the progenitor cells are transformed into cells containing detectable marker gene sequences. Claim No. Method 3. 5. 2. The method of claim 1, wherein said desired cell is a non-fungal plant cell. 6. 2. The method of claim 1, wherein said desired cells are animal cells. 7. 7. The method of claim 6, wherein the animal cell is a somatic cell. 8. The animal cells may be derived from chicken, mouse, rat, hamster, rabbit, sheep, or goat. A group consisting of animals, fish, pigs, cows or bulls, non-human primates, and humans. 8. The method of claim 7, wherein the cell is an animal selected from: 9. 7. The method of claim 6, wherein the animal cell is a pluripotent cell. 10. The animal cell may be chicken, mouse, rat, hamster, rabbit, sheep, From the group consisting of goats, fish, pigs, cows or bulls, and non-human primates 10. The method of claim 9, wherein the cells are of a selected animal. 11. 10. The method of claim 9, wherein the pluripotent cells are embryonic stem cells. 12. the desired gene sequence is essentially homologous to the predetermined gene sequence of the progenitor cell; The method according to any one of claims 1 to 3, which is the same. 13. A claim that the desired gene sequence is an analog of the predetermined sequence of the progenitor cell. The method of item 12 within the scope of. 14. the desired gene sequence is a human analog of the predetermined sequence of the progenitor cell; The method of claim 12. 15. A claim that the desired cell is a non-human cell expressing the desired gene sequence. The method of item 12 within the scope of. 16. If the desired gene sequence is a hormone, immunoglobulin, receptor molecule, Coating proteins selected from the group consisting of receptor molecule ligands and enzymes. 13. The method of claim 12. 17. A non-fungal plant containing an introduced recombinant DNA molecule containing the desired gene sequence. a physical cell, wherein the desired gene sequence is a predetermined gene sequence of the genome of the cell. Homologous regions (plurality) sufficient to undergo homologous recombination are adjacent to the desired gene sequence. Non-fungal plant cells in contact. 18. non-human containing an introduced recombinant DNA molecule containing the desired gene sequence an animal cell in which the desired gene sequence is a predetermined gene sequence in the genome of the cell. The desired gene sequence has sufficient homologous regions to undergo homologous recombination with the desired gene sequence. Non-human animal cells adjacent to. 19. A place created by any of the methods set forth in claims 1 to 3. Desired cells. 20. A desired cell produced by the method of claim 11. 21. A desired cell produced by the method of claim 12. 22. A chimera containing a cell derived from the desired cell of claim 19 A non-human animal that is an animal or a transformed animal. 23. the animal and the desired cell belong to the same species, and the species is chicken, mouse, rat, etc. Cat, hamster, rabbit, sheep, goat, fish, pig, cow or bull, or and a non-human primate according to claim 22. animals. 24. A chimera containing a cell derived from the desired cell of claim 20 A non-human animal that is an animal or a transformed animal. 25. The requested animal and the desired cell belong to the same species, and the species is chicken, mouse, rat, etc. Cat, hamster, rabbit, sheep, goat, fish, pig, cow or bull, or and non-human primates according to claim 24. animals. 26. A cell derived from the desired cell of claim 21 or a line thereof. A non-human animal that is a chimeric animal or a transformed animal. 27. the animal and the desired cell belong to the same species, and the species is chicken, mouse, rat, etc. Cat, hamster, rabbit, sheep, goat, fish, pig, cow or bull, or and non-human primates according to claim 26. animals. 28. Contains a cell derived from the desired cell of claim 5 or a line thereof A non-fungal plant that is a chimeric plant or a transformed plant. 29. Introducing the desired non-selectable gene sequence into the receptor requiring gene treatment. A method of gene treatment comprising: A. the desired non-selectable gene sequence; a DNA molecule containing a sequence of the desired gene sequence in a precursor cell of the receptor; the desired gene sequence sufficient to undergo homologous recombination with the intended gene sequence present in the An effective amount of a DNA molecule further containing two homologous regions flanking the gene sequence is B. C. enabling introduction of said DNA molecule into said precursor cell; Applicable The introduced DNA molecule is compatible with the planned gene sequence of the genome of the precursor cell. By undergoing homologous recombination, the desired unselectable gene sequence is Create a desired cell that has been inserted into the specified gene sequence, and inject the receptor into the cell. The presence or expression of the introduced gene sequence in the cell constitutes gene therapy. A method consisting of 30. 30. The method of claim 29, wherein said receptor is a non-fungal plant. 31. 30. The method of claim 29, wherein said receptor is an animal. 32. The animal is a chicken, mouse, rat, hamster, rabbit, sheep, or goat. , fish, pigs, cows or bulls, non-human primates, and humans? 32. The method of claim 31, wherein the method is selected from: 33. 33. The method of claim 32, wherein said animal is a human. 34. A desired non-selectable gene inserted within a predetermined gene sequence in the genome of a cell. A method for obtaining a desired animal cell or non-fungal plant cell containing a genetic sequence, the method comprising: A. The progenitor cells are subjected to the following i) under non-selective culture conditions or under first selective culture conditions: and ii) is incubated with a DNA molecule containing the gene sequence of [i) said desired a non-selectable gene sequence (wherein the DNA molecule is a precursor to the desired gene sequence) sufficient to cause homologous recombination of the genome of a somatic cell with the predetermined gene sequence. further containing two homologous regions flanking the desired gene sequence), and ii) a selectable gene sequence, the presence or expression of which in said progenitor cell; selecting the cells by culturing the cells under the first selective culture conditions; and its presence or expression in the precursor cells is determined by a second selective culture. Gene sequences that can be negatively selected by culturing the cells under Column] B. C. enabling introduction of said DNA molecule into said precursor cell; The introduced DN A molecule undergoes homologous recombination with the predetermined gene sequence of the genome of the progenitor cell. by causing the desired non-selectable gene sequence to become the predetermined gene sequence. Create the desired cells inserted into the column;D. under the first selective culture conditions. the cells are cultured and then allowed to undergo intrachromosomal recombination under non-selective culture conditions. by incubating the cells under the second selective culture conditions. and collecting the desired cells. 35. the cell is HPRT enzyme deficient and the selectable gene sequence is active PRT enzyme is expressed, and the first selective culture conditions are such that the PRT enzyme is present in the cells in order to grow. Incubating the cells under conditions requiring the presence of active HPRT enzyme The second selective culture conditions are such that the second selective culture conditions require activity within the cells in order to grow. Incubating the cells under conditions requiring the absence of active HPRT enzymes. 35. The method of claim 34, comprising: 36. the cell is APRT enzyme deficient and the selectable gene sequence is an active A PRT enzyme is expressed, and the first selective culture conditions are such that the PRT enzyme is present in the cells in order to grow. Incubating the cells under conditions requiring the presence of active APRT enzyme The second selective culture conditions are such that the second selective culture conditions require activity within the cells in order to grow. Incubating the cells under conditions requiring the absence of active APRT enzyme. 35. The method of claim 34, comprising: 37. the cell is TK enzyme deficient and the selectable gene sequence is an active TK enzyme The first selective culture conditions require active T in the cells to grow. by incubating the cells under conditions requiring the presence of the K enzyme. and the second selective culture conditions require active TK enzyme in the cells for growth. consisting of incubating said cells under conditions requiring the absence of The method of claim 34.