CA2084774A1 - Method for homologous recombination in animal and plant cells - Google Patents

Abstract

A method for producing animal cells which contain a desired gene sequence which has been inserted into a predetermined gene sequence by homologous recombination. The method permits the production of animal cells which have subtle and precise modifications of gene sequence and expression.

Description

WO~/19~96 PCT/-S91/04006 7 TITLE OF THE INVENTION:
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2 a~ P~t ~llc ÇROSS-REFERENCE ~O REL~TED ~PPLIC~TIONS

18 This application is a continuation-in-part application 19 of U.S. Patent Application Serial No. 07/537,458, filed on June 14, 1990.

22 FIELD OF THE INVENTION~
23 ::
24 The invention is directed toward recombinant DNA
technology, and more specifically, toward methods for 26 modifying endogenous genes in a chimeric or transgenic i7 animal or plant. The invention further pertains to the 28 animals/plants produced through application of the method, 29 and to the use of the method in medicine and agriculture.
This invention was supported by Government funds. The 31 Government has certain rights in this invention.

33 B~C~RO~D OF T~E INVENT~ON: -I. Chimeric nnd Tran~genic A~imal~

37 Recent advances in recombinant DNA and genetic 38 technologies have made it possible to introduce and express 39 a de~ired gene sequence in a recipient animal. Through the usc o~ such rethods, animals have been engineered to contain -`

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Wo 9l/19796 PCr/~'S91/0~006 gene sequences that are not normally or naturally present in 2 an unaltered animal. The techniques have also beer~ used to 3 produce animals which exhibit altered expression of 4 naturally present gene sequences.
The animals produced through the use of these me~hods 6 are known as either "chi~neric" or "transger. c" animals. In 7 a "chimeric" animal, only some of the animal's cells contain 8 and express the introduced gene sequence, whereas other 9 cells have been unaltered. The capacity of a chimeric animal to transmit the introduced gene sequenc~ to its 11 progeny depends upon whether the introduced gene sequences 12 are present in the germ cells of the animal. Thus, only 13 certain chimeric animals can pass along the desired gene 14 sequence to their progeny.
In contrast, all of the cells of a "transgenic" animal 16 contain the introduced gene sequence. Consequently, a 17 transgenic animal is capable of transmitting the introduced 18 gene sequence to its progeny.

II. Produ~tiou of TrzL~sgenic A~imal~:
21 Microinjection Method~

23 The most widely used method through which transgenic 24 animals have been produced involves in~ecting a DNA molecule into the male pronucleus of a fertilized egg (Brinster, R.L.
26 et al., Cell 27:223 (1981); Costantini, F. et al., Nature 27 294:92 ~1981); Harbers, K. et al., Nature 293:540 (1981);
28 Wagner, E.F. et al., Proc. Natl. Acad. Sc _LU~s-A-) 78:5016 29 (1981); Gordon, J.W. et al., Proc. Natl. Acad. Sci.~U.S.A.j 3~ 73:1260 (1976)).

31 The gene sequence being introduced need not be incor-32 porated into any kind of self-replicating plasmid or virus 33 (Jaenisch, R., Science, 240:1468-1474 (1988)). Indeed, the ! ' ; , , ', ' ' ' , ' ' . " .. ' ~ . , . .

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1 presence of vector DNA has been found, in many cases, to be 2 undesirable (Hammer, R.E. et al., Science 235:53 (1987);
3 Chada, X. et_al., Nature 319:685 (1986); Kollias, G. et al., 4 Cell 46:89 (1936); Shani, M., Molec. Cell. Biol. 6:2624 (1986); Chada, K. et al., Nature 314:377 (1985); Townes, T.
6 et al., EMBO J. 4:171S (1985)).
7 After being injected into the recipient fertilized egg, 8 the DNA molecules are believed to recombine with one another 9 to form extended head-to-tail concatemers. It has been propo3ed that such concatemers occur at sites of double-11 stranded DNA breaks at random sites in the egg's 12 chromosomes, and that the concatemers are inserted and 13 integrated into such sites (Brinster, R.L. et_al., Proc.
14 Natl. Acad. Sci. LU.S.A.) 82:4438 (1985)). Although it is, thus, possible for the injected DNA molecules to be 16 incorporated at se~eral sites within the chromosomes of the 17 fertilized egg, in most instances, only a single $ite of 18 insertion is observed ~Jaenisch, R., Science, 240:1468-1474 19 (1988); Meade, H. et al. (U.S. Patent 4,873,316)).
Once the DNA molecule has been injected into the 21 ~ertilized egg cell, the cell is implanted into the uterus 22 of a recipient female, and a:Llowed to develop into an 23 animal. Since all of the animal's cells are derived from 24 the implanted fertilized egg, all of the cells of ~he resulting animal (including the germ line cells) shall 26 contain the introduced gene sequence. If, as occurs in 27 about 30% of events, the first cellular division occurs 28 befora the introduced gene sequence has integrated into the 29 cell's genome, the resulting animal will be a chimeric animal.

31 By breeding and inbreeding such animals, it has been 32 possible to produce heterozygous and homozygous transgenic 33 animals. Despite any unpredicta~ility in the formation of , . ., , .; . .. . , , , , ~ ..

W~91t19796 PCT/-S91/04006 1 such transgenic animals, the animals have generally been 2 found to be stable, and to be capable of producing offspring 3 which retain and express the introduced gene sequence.
4 Since microinjection causes the injected DNA to be incorporated into the genome of the fertilized egg through 6 a process involving the disruption and alteration of the 7 nucleotide sequence in the chromosome of the egg at the 8 insertion site, it has been observed to result in the 9 alteration, disruption, or loss of function of the endo~enous egg gene in which the injected DNA is inserted.
11 Moreover, substantial alterations (deletions, duplications, 12 rearrangements, and translocations) of the endogenous egg 13 sequences flanking the inserted DNA have been obser~ed 14 (Mahon, K.A. et al., Proc. Natl. Acad. Sci. ru.s.A.L-85:ll65 is (1988); Covarrubias, Y. et al., Proc. Natl. Acad. Sci.
16 ~U.S.A.l 83:6020 (1986); Mark, W. et al., Cold S~r. ~arb.
17 Sym~. ~uant. Biol. 50:453 (1985)). Indeed, lethal mutations 18 or gross morphological abnormalities ha~e been observed 19 (Jaenisch, R., Science 240:146~-1474 (1988); First, N.L. et al., Amer. Meat_Sci. Assn. 39th Reciprocal Meat Conf. 39:41 21 (1986))).
22 Significantly, it has been observed that even if the 23 desired gene sequence of the microinjected DNA molecule is 24 one that is naturally found in the recipient egg's genome, integration of the desired gene seguence rarely occurs at 26 the site of the natural gene (Brinster, R.L. et al., Proc.
27 ~atl. Acad. Sci. (U.S.A.) 86:7087-7091 (1989)). Moreover, 28 introduction of the desired yene sequence does nat generally 29 alter the sequence of the originally preser.t egg gene.
Although the site in the fertilized egg's geno~e into 31 which the injected DNA ultimately integrates cannot be 32 predetermined, it is possible to control the expression of 33 the desired gene sequence such that, in the animal, ~ .

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1 expression of the sequence will occur in an organ or tissue 2 specific manner (reviewed by Westphal, H., FASEB J. 3:117 3 (1989); Jaenisch, X., Science 240:1468-1474 tl988)).
4 The success rate for producing transgenic animals is greatest in mice. Approximately 25% of fertilized mouse 6 eggs into which DNA has been injected, and which have been 7 implanted in a female, will become transgenic mice. A lower 8 rate has been thus far achieved with rabbits, sheep, cattle, 9 and pigs (Jaenisch, R., Science 240:1468-1474 (1988);
Hammer, R.E. et al., J. Animal. Sci. 63:269 (1986); Hammer, 11 ~.E. et al., Nature 315-680 (19%5); Wagner, T.E. et al., 12 Therioqenoloav 21:29 (1984)). The lower rate may reflect 13 greater familiarity with the mouse as a genetic system, or 14 may reflect the difficulty of visualizing the male pronucleus of the fertilized eggs of many farm animals 16 (Wagner, T.E. et_al., Therioaenoloqv 21:29 (1984)).
17 Thus, the productian of transgenic animals by 18 microinjection of DNA su~fers from at least two major 19 drawbacks. First, it can be accomplished only during the single-cell stage of an animal's life. Second, it requires 21 the disruption of the natural sequence of the DNA, and thus 22 is often mutagenic or teratogenic (Gridley, T. et al., 23 Trends Genet. 3:162 (1987)3.

III. Produetion of Chimeric ,~nd Transgenic Animals:
26 Recombinant Vir~l nnd Retroviral ~ethod~
~7 28 Chimeric and transgenic animals may also, be produced 29 using recombinant viral ar retroviral techniques in which the gene sequence is introduced into an animal at a multi-31 cell stage. In such methods, the desired gene sequence is 32 introduced into a virus or retrovirus. Cells which are 33 in~ected with the virus acquire the introduced gene .. .. . ... ... . . .

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l sequence. If the virus or retrovirus infects every cell of 2 the animal, then the method results in the production of a 3 transgenic animal. If, however, the vlrus infects only some 4 of the animal's cells, then a chimeric animal is produced.
S The general advantage of viral or retroviral methods of 6 producing transgenic animals oYer those methods which 7 involve the microinjection of non-replicating DNA, is that 8 it is not necessary to perform the genetic manipulations at 9 a single cell stage. Moreover, infection is a highly efficient means for introducing the DNA into a desired cell.
11 Recombinant retroviral methods for producing chimeric or 12 transgenic animals ha~e the advantage that retroviruses 13 integrate into a host's genome in a precise manner, 14 resulting generally in the presence of only a single integrated retrovirus (although multiple insertions may 16 occur). Rearrangements of the host chromosome at the site 17 of integration are, in general, limited to minor deletions 18 (Jaenisch, R., Science 240:1468-1474 tl988); see also, 19 VE.rmUS~ H., In: RNA Tumor Viruses (Weiss, R. et al., Eds.), Cold Spring Harbor Press, Cold Spring Harbor, NY, pp. 369-21 Sl2 (1982)). The method is, however, as mutagenic as micro~
22 injection methods.
23 Chimeric animals have, for example, been produced by 24 incorporating a desired gene sequence into a virUs (such as bovine papilloma virus or polyoma) w~ich is capable of 26 infecting the cells of a host animal. Upon infection, the 27 virus can be maintained in an infected cell as an 28 extrac~romosomal episome (Elbrecht, A. et al., Molec. Cell.
29 iol~ 7:1276 (1987); Lacey, M. et al., Nature 322:609 (1986); Leopold, P. et al., Cell 51:885 (1987)). Although 31 this method decreases the ~utagenic nature uf 32 chimeric/transgenic animal ~ormation, it does so by 33 decreasing germ line stability, and increasing oncogenicity '~,' '.

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1 Pluripotent embryonic stem cells (referred to as "ES"
2 cells) are cells which may be obtained from embryos until 3 the early post-implantation stage of embryogenesis. The 4 cells may be pr~pagated in culture, and are able to differentiate either in vitro or in vivo upon implantation 6 into a ~ouse as a tumor. ES cells have a normal Xaryotype 7 ~Evans, M.J. et al., Nature 292:154-156 (1981); Martin, G.R.
8 et al., Proc. Natl. Acad. Sci. (U.S.A.) 78:7634-7638 9 (1981)).
Upon injection into a blastocyst of a developing embryo, 11 ES cells will proliferate and differentiate, thus resulting 12 in the production of a chimeric animal. ES cells are 13 capable of colonizing both the somatic and germ-line 14 lineages of such a chimeric animal (Robertson, E. et al~
15 Cold S~rin~ Harb. Conf. Cell_Prolif. 10:647-663 (1983);
16 Bradley A. et al., Nature 309:255-256 (1984); Bradley, A. et 17 al., Curr. Top. Devel. Biol. 20:357-371 (lg86); Wagner, E.F.
18 et al., Cold SPrina Harb. SYm~. Quant. Biol. 50:691-700 19 (1985); (a:Ll of which references are incorporated herein by 20 reference).
21 In this method, ES cells are cultured in vitro, and 22 infected with a viral or retroviral vector containing the 23 gene sequence of interest. Chimeric animals generated with 24 retroviral vectors have been found to have germ cells which 25 either lack the introduced gene sequence, or contain the 26 introduced sequence but lack the capacity to produce progeny 27 cells capable of expressing the introduced sequence (Evans, -28 M.J. et al., Cold S~rinq Harb. S~ymP. ~uant Biol. 50:685-689 29 (1985); Stewart, C.L. et al., EMBO J. 4:3701-3709 (1985);
30 Robert~on, L. et al., Nature (1986); which references are 31 incorporated herein by reference).
32 Because ES cells may be propagated in vitro, it is 33 possible to manipulate such cells using the techni~ues of ~

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WO 91/19796 PCr/-S91/04006 l somatic cell genetics. Thus, it is possible to select ES
2 cells which carry mutations (such as in the hPrt gene 3 (encoding hypoxanthine phosphoribosyl transferase) (Hooper, 4 M. et al., Nature 326:292-295 (1987); Kuehn, M.R. et al., Nature 326:295-298 (1987)). Such selected cells can then be 6 used to produce chimeric or transgenic mice which fail t~
7 express an active HPRT enzyme, and thus provide animal 8 ~odels for diseases (such as the Lesch-Nyhan syndrome which 9 is characterized by an HPRT deficiency) (Doetschman, T. et al., Proc. Natl. Acad. Sci. (U.S.A.) 8S 8583-8587 (1988)).
ll As indicated above, it is possible to generate a 12 transgenic animal from a chimeric animal (whose germ line 13 cells con~ain the introduced gene sequence) by inbreeding.
14 The above-described methods permit one to screen for the desired genetic alteration prior to introducing the trans-16 fected ~S cells into the blastocyst. One drawback of these 17 methods, however, is the inability to control the site or 18 nature of the integration of the vector.
19 :'' IY. Productio~ of Chimeric ,~nd ~ransgenic Anim~ls:
21 Plasmi~ ~ethods -~

23 The inherent drawbacks of t:he above-described methods 24 for producing chimeric and transgenic animals have caused researchers to attempt to identify additional methods 26 through which such animals could be produced.
27 Gossler, A. et al., ~or example, have described the use 28 of a plasmid vector which had been modified to contain the 29 gene for neomycin phosphotransferase (nPtII gene) to transfect ES cells in culture. The presence of the nptII
31 gene conferred resistance to the antibiotic G418 to ES cells 32 that had been infected by the plasmi~d (Gossler, A. et_al., 33 ~roc. Natl. Acad. Sci. (U.S.A.) 83:9065-9069 (1986), which :

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WOgl/19~96 PCT/~S91/~4006 _9~ "P 74 1 reference is incorporated herein by reference). The 2 chimeric animals which received the plasmid and which became 3 resistant to G418, were found to have integrated the vector 4 into their chromosomes. Takahashi, Y. et al. have described the use of a plasmid to produce chimeric mice 6 cells which expressed an avian crystallin gene (DeveloPment 7 102:258-269 (1988), incorporated herein by re~erence). The 8 avian gene was incorporated into a plasmid which contained 9 the nPtII gene. Resulting chimeric animals were found to express the avian gene.

12 V. I~troduction of Ge~e sequenreq anto ~omatic Cells 14 DNA has been introduced into somatic cells to produce variant cell lines. hprt-deficient Chinese hamster ovary 16 ~CHO) cells have been transformed with the CHO hPrt gene in 17 order to produce a prototrophic cell line ~Graf, L.H. et 18 al., Somat. Cell Genet. S:1031-1044 (1979)). Folger et al.
19 examined the fate of a thymidine kinase gene (tk gene) which had been microinjected into the nuclei of cultured mammalian 21 cells. Recipient cells were found to contain from 1 to lO0 22 copies of the introduced gene sequence integrated as 23 concatemers at one or a few sites in the cellular genome 24 (Folger, K.R. et al., ~olec. Cell. _Biol. 2:1372-1387 (1982)). DNA mediated transfo~mation of an ~NA polymerase 26 II gene into Syrian hamster cells has also been reported 27 (Ingles, C. et al., Molec Cell. Biol. 2:666-673 (1982)).
28 Plasmids conferring host neomycin resistance and 29 guanosine phosphotransferase activity have been transfected into Chinese hamster ovary cells to generatP novel cell 3~ lines (Robson, C.N. et al., Mutat. Res. 163:201-208 (1986)).

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WO91/19796 PCT/~IS91/04006 t )~ 1 0 1 VI. C~imeric or Trnnsge~ic Pl~ntq 3Extensive progress has been made in recent years in the 4fields of plant cell genetics and gene technology. For many 5genera of plants, protoplast regeneration techniques can be 6used to regenerate a plant from a single cell (Friedt, W. et 7al. Proq. Botanv 49:192-215 (1987~; Brunold, C. et al., 8Molec. Gen. Genet. 208:469 473 (1987); Durand, J. et al., 9Plant _ Sci. 62:263-272 (1989) which references are 10incorporlted herein by re~erence).
11Several methods can be used to deliver and express a 12foreign gene into a plant cell. The most widely used method 13employs cloning the desired gene sequence into the Ti 14plasmid of the soil bacterium A. tumori~aciens (~omari, T.
15et al., J. Bacteriol. 166:88-94 (1986); Czako, M. et al., 16Plant Moi. ~3iol. 6:101-109 t1986); Jones, J.D.G. et al., ~ -17EMBO J. 4:2411-2418 (1985); Shahin, E.A. et al., Theor.
18AP~1. Genet. 73:164-169 (1986)). The frequency of 19transformation may be as high as 70%, depending upon the '~
20type of plant used (Friedt, W. gt al. Pro~_8Otanv 49:192-21215 (1987)).
22Plant viruses have also been exploited as vectors for 23the delivery and expression of foreign genes in plants. The 24cauliflower mosaic virus (Brisson, N. et al., Nature 25310:511-514 (1984) has been particularly useful for this 26purpose (Shah, D.M. et al., Science 233:478-481 (1986);
27Shew~aker, C.K. et al., Virol. 140:281-288 (1985). Vectors 28have also been prepared from derivatives of RNA viruses 29(French, R. et al., Science 231:1294-1297 ~1986).
30Techniques of microinjection (Crossway, A. et al., 31Molec. Gen. Genet. 202:179-185 (1986); Potrykus, I. et al., 32Molec. Gen. &enet. 199:169 177 (1985)), have been ~sed to 33accomplish the direct transfer of gene sequences into plant ;;~
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1 cells. Transformation with a plasmid capable of site 2 specific recombination has bee~ used to introduce gene 3 sequences into As~erqillus (May, G.S., J. Cell Biol.
4 109:2267-2274 (1989); which reference is incorporated herein by reference).
6 Electroporation has been identified as a method for 7 introducing DNA into plant cells (Fro~m, M.E., et al., Proc.
8 Natl. Acad. sci. (U.S.A.) 8~:5824-5828 (1985); Fromm, M.E.
9 ~t_aL., Nature 319:?91-793 (1986); Morikawa, H. et al., Gene 41:121-124 (1986); Langridge, W.H.R. et al., Theor. Appl.
11 Genet. 67:443-455 (1984)).
12 Gross genetic mutations can be produced in plant cells 13 using transposable elements (Saedler, H. et al., EMBO_J.
14 4:S85-590 (1985); Peterson, P.A., BioEssavs 3:199-204 (1985))- Such elements can initiate chromosomal 16 rearrangements, insertions, duplications, deletions, etc.
17 Chimeric plants can be regenerated from such cells using the 18 procedures described above.
19 A major deficiency of present methods for gene manipulation in plants is the difficulty of selecting the 21 desired recombinant cell (Bruno:Ld, C. et al., Molec Gen.
22 Genet. 208:469-473 (1987)). In an attempt to address this 23 deficiency, kanamycin resistance and nitrate reductase 24 deficiency have been used as selectable markers (Brunold, C.
et al., Molec. Gen. Genet. 208:469-473 (1987)).
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WO91/19796 P~T/~S91/04006 ~ '~`J'~ 12-l VII. Conclu~ions 3 The application of the above-described technologies has 4 the potential to produce types of plants and animals which cannot be produced through classical genetics. For example, 6 animals can be produced which suffer from human diseases 7 (such as AIDS, diabetes, cancer, etc.), and may be valuable 8 in elucidating therapies for such diseases. Chimeric and 9 transgenic plants and animals have substan~i~l use as probes of natural gene expression. When applied to liv~stock and ll food crops, the technologies have the potential of yielding 12 improved food, fiber, etc.
13 Despite the successes of the above-described techniques, 14 a method for producing chimeric or transgenic plants and animals which was less mutagenic, and which would permit 16 defined, specific, and delicate manipulation of the inserted ~7 gene sequence at a specific chromosomal location would be 18 highly desirable. ~
19 .': , 21 ~R~EF DE~8CRIPTION OF T~IE FIGIJR.E9 2 2 :~
23 Figure l illustrates t~e use of replacement vectors and 24 insertion vectors in gene targeting. Figure lA is a diagrammatical representation of the use of a replacement ~
26 vector in yene targeting; Figure lB illustrates the use of ~ -27 an insertion vector to produce subtle mutations in a desired ~ .
28 gene sequence.
29 Figure 2 is a diagra~matical representation of a DNA
molecule which has a region of heterology located at a 31 proposed insertion site. Figure 2A shows a construct with a 32 2 kb region of heterology. Figure 2B shows a construct with 33 a 26 base long region of he~erology which has been ' ;.
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l linearized at the center of the region of heterology.
2 Figure 2C shows a construct with a region of heterology 3 located internal to the re~ion of homology at which 4 recombination is desired. In Figure 2c, the normal BamHI
site of the vector has been changed to an NheI site and the 6 normal EcoRI site of the vector has been changed to a BamHI
7 site. The vector is linearized with XhoI
8 Figure 3 is a diagrammatical representation of the 9 mechanism through which a "humanized" gene may be introduced into a chromosomal gene sequence in a one step ~ethod.
11 Figure 4 is a diagrammatical representation of the 12 mechanism through which a large qene may be introduced into 13 a chromosomal gene sequence so as to place the gene under 14 the transcriptional control of a heterologous promoter (for example, to place a human gene under the control of a mouse 16 gene). The first step is additive and the !second is a 17 replacement event. Fiyure 4A shows the first step of the 18 process; Figure 4B shows the second step of the process.
l9 The repair recombination event may be configured to remove all of the mouse coding exons if desired. ~ !`
21 ~ Figure 5 is a diagramatical representation of the use of ~-22 a positive selection/ negativle selection "cassette" to ;
23 introduce subtle mutations into a chromosome.
24 Figure 6 is a diagrammatical representation of a multi-step method (~igures 6A-6E) for introducing small or large 26 desired gene sequences into a contiguous region o$ a cellis 27 genome. The figure illustrates a vector capable of 28 facilitating the sequential addition of overlapping clones 29 to construc~ a large locus. Every step i9 selectable. ~;
Subsequent additions may be made by returning to steps 4 and 31 5 as ~any times as required, selecting for insertion in HAT
32 medium, and for repair in media supplemented with 6 : . -: .
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wu~ J~ PCT/~S91/0~006 1 thioguanine. This procedure may also be accomplished at the 2 other end of the locus if required.
3 Figure 7 is a diagrammatical reprPsentation of the 4 vectors used in a co~electroporation experiment to mutate the hprt gene.
6 Figure 8 illustrates the predicted structure ~f the _~E~
7 gene following homologous recombination of the IV6.8 vector.
8 ER is the predicted size fragment indicative of the 9 homologous recombination event. End, ~ is the endogenous fragment, duplicated by the recombination event. End is the 11 predicted flanking fragment detected by the partial cDNA
12 probe used in these experiments.
13 Figure 9 shows the reversion of homologous recombinants 14 generated with insertion vectors.
Figure 10 illustrates the use of Poly A selection as a 16 means for selecting homologous recombination events. ~
17 Figure 11 illustrates the use of the. invention to -18 introduce insertions into ~he sequence of a desired gene of 19 a cell. Figure llA is a diagram of the c-src locus showing relevant restriction sites (E=EcoRI; N~coI; X=XhoI;
21 H=HindIII; ~=BamHI; Nh=NheI). Figure llB illu~trates the ~-22 src 14 vector used to introduce mutations into the c-src 23 locus; Figure llC illustrates the subtle mutation introduced 24 through the use of this vector. ~-Figure 12 illustrates the use of the invention to 26 introduce substitutions into the ~equence of a desired gene 27 of a cell. Figure 12A is a diagram of the c-src locus 28 showing relevant restriction sites (E=EcoRI; N=NcoI; X=XhoI;
29 H=HindIII; B=9amHI; Nh-NheI). Figure 12B illustrates the src 33 vector used to introduce mutations into the c-src 31 locus; Figure 12C illustrates the subtle mutatio~ introduced 32 through the use of this vector.

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WO 91/1979~ PCl/l S91/041~06 --15-- ~ r ~r 4 1 Figure 13 illustrates a comparison between targeted and 2 random recombinatlonal events. In a random recombinational 3 event, although concatemers can e~cise duplications, one 4 copy of the vector must remain in the genome. In contrast, in a targeted recombinational event, all sequences, except 6 the desired sequence is excised from the genome.
8 ~MM~Ry OF T~E INYENTION:

10The present invention provides a method for obtaining a 11desired animal or non-fungal plant cell which contains a 12predefined, specific and desired alteration in its g~nome.
13~he invention further pertains to the non-human animals and 14plants which may be produced from such cells. The invention 15additionally pertains to the use of such non-human animals 16and plants, and their progeny in research, medicine, and I7agriculture.
~8In detail, the invention provides a method for obtaining 19a desired animal or non-fungal plant cell which contains a 20desired non-selectable gene sequence inserted within a 21predetermined gene sequence of the cell's genome, which 2~method comprises:
23A. incubating a prPcurr,or cell with a DNA molecule 24containing the desired non-selectable gene sequence, wherein 25the DN~ molecule additionally contains two regions of 26homology which flank the desired gene sequence, and which 27are sufficient to permit the desired gene seguence to 28undergo homologous recombination with the predetermined gene 29sequence of the genome of the precursor cell;
3~~. causing the DNA molecule to be introduced into 31the precursor cell;
32C. permitting the introduced DNA molecule to 33undergo homologous recombination with the predetermined gene :.,: - ,. :

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1 sequence of the genome of the precursor cell t3 thereby 2 produce the desired cell wherein the desired non-selectable ~ gene sequence has been inserted into the predeter~ined gene 4 sequence; and D. recoYering the desired cell.
6 The invention further includes the embodiments of the 7 above-described method wherein the DNA molecule contains a 8 detectable marker gene sequence, and/or wherein the DNA
9 molecule is introduced into the precursor cell by subjecting the precursor oell and the DNA molecule to electroporation 11 (especially wherein in step ~, the precursor cell is 12 si~ultaneously subjected ~o electroporation with a second 13 DNA molecule, the second DNA molecule containing a 14 detectable marker gene sequence).
The invention further includes the embodiments of the 16 above-described method wherein the desired cell is a non~
17 fungal plant cell, a somatic animal cell (especially one 18 selected from the group consisting of a chicken, a mouse, a 19 rat, a hamster, a rabbit, a sheep, a goat, a fish, a pig, a cow or bull, a non-human primate and a human), a pluripotent 21 animal cell (especially one selected from the group 22 consisting of a chicken, a mouse, a rat, a hamster, a 23 rabbit, a sheep, a goat, a fish, a pig, a cow or bull, and 24 a non-human primate). The invention includes with the embodiment wherein the pluripotent cell is an embryonic stem 26 cell.
27 The invention also includes the embodi~ents of the 28 above described methods wherein the desired gene sequence is 29 subst ntially homologous to the predetermined gene ee~uence of the precursor cell and/or wherein the desired gene 31 sequence is an analog (and especially a human analog) of the 32 predetermined sequence of the precursor cell.

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WO91/19796 p~r/~s9l/o4oo6 -17- ~ c i ~

1The lnvention also includes the e~bodiment wherein the 2desired gene sequence encodes a protein selected from the 3group consisti~g of: a hormone, an immunoglobulin, a 4receptor molecule, a ligand of a rec2ptor molecule, and an 5enzyme.
6The invention also includes a non-fungal plant cell 7which contains an introduced recombinant DNA molecule 8containing a desired gene sequence, the desired gene 9sequence being flanked by regions of homology which are 10su~fi~ient to permit the desired gene sequence to undergo 11homologous recombination with a predetermined gene sequence 12of the genome of the cell.
13The invention also includes a non-human animal cell 14which contains an introduced recombinant DNA molecule 15containing a desired gene sequence, the desired gene 16sequence being flanked by regions of homolo~y which are 17sufficient to permit the desired gene sequence to u~dergo 18homologous recombination with a predetermined gene sequence 19of the genome of the cell.
20The invention also includes 1:he desired cell produced by 21any of the above-described methods.
22The invention also includes a non-human animal 23containing a cell derived~from the above-described desired -~
24cell, or a descendant thereof, wherein the animal is either 25a chimeric or a transgenic animal, and particularly includes 26the embodiment wherein the non-h~man animal and the desired 27cell are of the same species, and wherein the species is ;
28selected from the group consisting of: a chicken, a mouse, 29a rat, a ha~ster, a rabbit, a sheep, a goat, a fis~, a pig, 30a cow or bull, and a non-human primate.
31The invention also includes a non-fungal plant 32containing a cell derived from the above-described desired - : . . - .

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WOgl/19796 PCT/~591/04~06 1non-fungal plant cell, wherein said non-fungal plant is 2either a chimeric or a transgenic plant.
~The invention also includes a method of gene therapy 4which comprises introducing to a reclpient in need of such 5therapy, a desired non-selectable gene sequence, the method 6comprising:
7A. providing to the recipient an effective amount 8of a DNA molecule containing the desixed non-selectable gene 9sequence, wherein the D~A molecul~ additionally contains two 10regions of homology which flank the desired gene sequence, ~1and which are sufficient to permit the desired gene seguence 12to undergo homologous recombination with a predetermined 13gene sequence present in a precursor cell of the recipient;
14B. permitting the DNA molecule to be introduced 15into the precursor cell;
16C. permitting the introduced DNA molecule to 17undergo homologous recombination with the predetermined gene 18se~uence of the genome of the precursor cell to thereby 19produce a desired cell wherein the desired non-selectable 20gene sequence has been inserted into the predetermined gene 21sequence; and wherein the presence or expression of the 22introduced gene sequence in the cell of the recipient 23comprises the gene therapy;
24In particular, the invention includes the embodiments of 25the above-stated method wherein the recipient is a non-26fungal plant, or a human or a non-human animal (particularly 27a non-human animal is selected from the group consisting of~
28a chicken, a mouse, a rat~ a hamster, a rabbit, a sheepj a 29goat, a fish, a pig, a cow or bull, a non-hu~an primate and 30a human).

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WO 91/19796 PCI/~S9~/04006 r r J ~c 1The lnvention also provides a method for obtaining a 2desired animal or non-fungal plant cell which contains a 3desired non-selectable gene sequence inserted within a 4predetermined gene sequence of the cell's geno~e, which 5method comprises:
6A. incubating a precursor cell under non-selective 7culture conditions, or under a first set of selective 8culture conditions, with a DNA molecule containing:
9i) the desired -non-selectable gene seguence, 10wherein the DNA molecule additionally contains 11two regions of homology which flank the desired 12gene sequence, and which are sufficient to 13permit the desired gene sequence to un~ergo 14homologous recombination with the predeterminçd 15gene sequence of the genome of the precursor 16cell; and 17ii~ a selectable gene sequence whose presence or 18expression in the cell can be selected for by 19cuituring the cells under the first set of 20selective culture conditions, and whose 21presence or expression in the cell can be 22selected against by culturing the cells under 23a second set of selective culture conditions;
24B. permitting the DNA molecule to be introduced 25into the precursor cell;
26C. permitting the introduced DNA molecule to 27undergo homologous recombination with the predetermined gene 28sequence of the genome of the precursor cell to thereby 29produce the desired cell wherein the desired non-selectable 30gene sequence has been inserted into the predetermined gene 31sequence; and 32D. recovering the desired cell by culturing the 33cell under the first set of selective culture conditions, by ''~

WO91/1979h PCr/~S91/04006 ~ t~ 20-l then permitting the cell to undergo intrachromosomal 2 recombination under non-selective culture conditions, and by 3 then incubating the cell under the second set of selective 4 culture ~onditions.
The invention also includes the embodiment wherein the 6 cell is deficient in an HPRT, APRT, or TK enzyme, and 7 wherein the sel~ctable gene sequence expresses an active 8 HPRT, APRT, or TX enzyme, and wherein the first set of 9 selective culture conditions comprises incubation of the cell under conditions in which ~he presence of an active 11 HPRT, APRT, or ~K enzyme in the cell is required for growth, 12 and wherein the second set of selective culture conditions 13 comprises incub~tion of the cell under conditions in which 14 th~ absence of an active HPRT, APRT, or TK enzyme in the ;~
cell is re~uired for growth.

18 D~8CRIiTION OF THE PREFER~ED EMBODIMENTS:
19 :
The present invention concerns a method for introducing 21 DNA into the genome of a recipi1ent plant or animal cell.
22 The method may be used to introduce such DNA into germ line 23 cells of animals (especially, rodents (i.e. mouse, rat, 24 hamster, etc.), rabbits, sh ep, goats, fish, pigs, cattle and non-human primates) in order to produce chimeric or 26 transgenic animals. The methods may also be used to 2i introduce DNA into plant cells which can then be manipulated `28 in order to produce chimeric or transgenic plants.
29 Alternatively, the method may be used to zlter the s~matic cells of an animal (including humans) or a plant.
31 The plants and plant cells which may be manipulated through 32 application of the disclosed method include all 33 multicellular, higher (i.e. non-fungal or non-yeast) plants.

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W ~ 91/19796 PCT/~591/04006 -2l-~ ~ ~J~. r 1 I. ~omologou~ Recombination 3The present invention provides a method for introducing 4a desired gene sequence into a plant or animal cell. Thus, 5it is capable of producing chimeric or transgenic plants and 6animals having defined, and specific, gene alterations.
7An understanding of the process of homologous 8recombination (Watson, J.D., In: Molecular ~loloqY of the 9Gene, 3rd Ed., W.A. Benj2min, Inc., Menlo Park, CA ~977), 10which reference is incorporated herein by reference) is 11desirable in order to fully appreciate the present 12invention.
13In brief, homologous recombination is a well-studied 14natural cellular process which results in the scission of 15two nucleic acid molecules having identical or substantially 16similar sequences (i.e. "homologousl'), and the ligation of 17the two molecules such that sne region of each initially 18present molecule is now ligatecl to a region of the other 19initially present molecule (';edivy, J.M., Bio-Technol.
206:1192-1196 (1988), which reference is incorporated herein 21by reference).
22Homologous reco~bination is, thus, a sequence specific 23process by which cèlls can transfer a "region" of DNA from 24one DNA molecule to another. As used herein, a "region" of 25DNA is intended to generally refer to any nucleic acid 26molecule. The region may be of any length from a single 27base to a sub-~tantial fragment of a chromosome.
28For homologous recombination to occur between two DNA
, ~ .
29molecules, the molecules must possess a "region of homology"

30with respect to one another. Such a region of homology must -31be at least two base pairs long. Two DNA ~olecules possess 32such a "règion of homology" when one contains a region whose ;

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1 sequence is so similar to a region in the second molecule 2 that homologous recombination can occur.
3 Recombination is catalyzed by enzymes which are 4 naturally present in both prokaryotic and eukaryotic cells.
S The transfer of a region of DNA may be envisioned as 6 occurring through a multi-step process.
7 If either of the two participant molPcules is a circular 8 molecule, then the above recombination event results in the -9 integration of the circular molecule into the other participant.
ll Importantly, if a partic~lar region is flanked by 12 regions of homology ~which may be the same, but are 13 preferably different), then two xecombinational events may 14 occur, and result in the exchange of a region of DNA between two DNA molecules. Recombination may be "reciprocal," and 16 thus results in an exchange of DNA regions between two 17 recombining DNA molecules. Alternatively, it may be "non~
18 reciprocal," (also referred to as "gene conversion") and 19 result in both recombining nucleic acid molecules having the same nucleotide sequence. There are no constraints 21 regarding the size or sequence of the reyion which is 22 exchanged in a two~event recombinational exchange.
23 The frequency of recombination between two D~A molecules 24 may be enhanced by treating the introduced DNA with agents which stimulate recombination. Examples of such agents 26 include tximethylpsoralen, W light, etc. ~;

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", : ,, WO 91/t97~6 PCr/l~S91/04006 - 2 3 ~ r 1II. Pro~uction of Chimeric and ~rA~qgenic Animal~:
2 Ge~e Targeting Method3 4 One approach to producing animals having defined and specific genetic alterations has used homologous 6 recom~ination to control the site of integration of an 7 introduced marker gene sequence in tumor cells and in 8 fusions between diploid human fibroblast and tPtraploid 9 mouse erythroleukemia cells (Smithi~s, O. et al., Nature 317:230-234 (1985)).
11 This approach was further exploited by Thomas, K. R., 12 and co-workers, who described a general method, known as 13 "gene targeting," for targeting mutations to a preselected, 14 desired gene sequence of an ES cell in order to produce a transgenic animal (Mansour, S.L. et al., Nature 336:348-352 16 (1988); Capecchi, M.R. Trends Genet. 5:70-76 (1989);
17 Capecchi, M.R. et al., In: Current_ communications in 18 Molecular Biolo~v, Capecchi, M.R. (ed.), Cold Spring Harbor 19 Press, Cold Spring Harbor, NY (1989), pp. 45-52, which references are incorporated herein by reference).
21 Gene targeting has been used to produce chimeric and 22 transgenic mice in which an nl~tII gene has been inserted 23 into the ~2-microglobulin locus (Xoller, B.H. et al., Proc.
24 Natl. Acad. Sci._(U.S.A.) 86;8932-8935 (1989); Zijlstra, ~.
et al., N_ture 342:435-438 (1989); Zijlstra, M. et al., 26 Nature 344:742-746 (1989); DeChiaba et al., ature 345:78-80 27 (1990)j. Similar experimen~s have enabled the production of 28 chimeric and transgenic ani~als having a c-abl ~ene which 29 has been disrupted by the insertion of an n~tII gene (Schwartz~erg, P.L. et al., Scie~ce ~ 799-803 ~19a9)~

31 The technigue has been used to produce chimeric mice in 32 which the en-2 gene has been disrupted by th~ insertion of .. . .. . . . . .

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W O 91/19796 PCT/~'S91/04006 ~t~ -24-1 an nE~II gene (Joyner, A~L. et al., Nature 338:153-155 2 (1989)).
3 Gene targeting has also been used to correct an hprt 4 deficiency in an hPrt ES cell line. Cells corrected of the deficiency were used to produce chimeric animals.
6 5ignificantly, all of the corrected cells exhibited gross 7 disruption of the regions flanking the h~rt locus; all of 8 the cells tested were found to contain at least one copy of 9 the vector used to correct the defici~ncy, integrated at the hDrt locus (Thompson, S. et al., Cell 56:313-321 (1989);
ll Xoller, B.H. et al., Proc. Natl. Acad. sci. (U.S.A.) 12 86:8927-8931 (1989)).
13 In order to utilize the "gene targeting" method, the 14 gene of interest must have been previously cloned, and the intron-exon boundaries determined. The method results in 16 the insertion of a marker gene (i.e. the n~tII gene) into a 17 translated region of a particular gene of interest. Thus, ~8 use of the gene targeting method results in the gross l9 destruction of the gene of interest.
Recently, chimeric mice carrying the homeobox hox 1.1 21 allele have been produced using a modification of the gene 22 targeting method (Zimmer, A. et: al., Nature 338:150-154 23 (1989). In this modification, the integration of vector 24 sequences was avoided by microinjecting ES cells with linear DNA containing only a portion of the hox 1.1 allele, without 26 any accompanying vector sequences. The DNA was found to 27 cause the gene conversion of the cellular hox allele.
28 Setection was not used to facilitate the recovery of the 29 "converted" ES cells, which were identified using the polymerase chain reaction ("PCR"). Approximately 50~ o~
31 cells which had been clonally purified from "converted"
32 cells were ~ound to contain the introduced hox 1.1 allele, 33 suggesting to Zimmer, A. et al. either chromosomal i, .
.

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WO91/197g6 PCT/~S9~/04006 1 instability or conta~ination o sa.ple. None of the 2 chimeric mice were found to be able to transmit the 3 "converted" gene to their progeny (Zimmer, A. et al., In:
4 Current Communications in Molecular ~ioloav, Capecchi, M.R.
(ed.), Cold Spring Harbor Press, Cold Spring Harbor, NY
6 (1989), pp. 53-58).
7 The use of the gene targeting method is illustrated in 8 Figure lA. In that figure, a gene construct is produced in 9 which the nptII gene is inserted into an exon (designated region "3") of a sequence of the hprt gene. The construct 11 is then permitted to undergo recombination with the hPrt 12 gene of a cell. Such reco~bination results in the 13 replacement of the exon 3 sequence of the cell with the 14 disrupted exon 3 - n~tII sequence of the construct.
Significantly, as illustrated in Fiyure lA, the use of gene 16 targeting to alter a gene of a cell results in the formation 17 of a gross alteration in the sequence of that gene. As 18 indicated in Figure lA, the efficiency of gene targeting is 19 approximately 1/300.
~ 0 . .~
21 III. Pro~uction of Chimeric ~nd Tr~n~genic An~m~
22 ~se of Insertion Vectoxs 24 In contrast to the above-described methods, the present invention is capable of producing subtle, precise, and 26 predetermined mutations in the sequence of a desired gene of ~;
27 a cell. The present invention has several embodiments, the 28 simplest of which is illustrated in Figure lB.
29 As shown in Figure lB, an insertion ~ector is used to -~
~utate the nucleotide sequence o~ the hprt gene. The use of ~ `
31 this vector type in combination with a second selectable 32 reversion event prevents the disruption of the chromosome by -3~ the n~tII gene or by the vector sequences. Thus, gross ~. -.

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WO91/19796 PCT/~'S91/04006 l distortions of the recipient chromosome are avoided by the 2 present invention. Moreover, the efficiency of the gene 3 targeting was substantially improved (i.e. l/32 as opposed 4 to l/300).
The DNA molecule(s) which are to be introduced into the 6 recipient cell preferably contains a region of homology with 7 a region of the cellular genome. In a preferred embodiment, 8 the DNA molecule will con~ain two regions of homology with 9 the genome (both chromosomal and episomal) of the pluripotent cell. These regions of homology will preferably ll flank a "desired gene sequence" whose incorporation into the 12 cellular genome is desired. As stated above, the regions of 13 homology may be of any size greater than two bases long.
14 Most preferably, the regions of homology will be greater than lO bases long.
16 The DN~ molecule(s) may be single stranded, but are 17 preferably dou~le stranded. The DNA molecule(s~ may be 18 introduced to the cell as one or more RNA molecules which l9 ~ay be converted to DNA by reverse transcriptase or by other means. Preferably, the DNA molecule will be double stranded 21 linear molecule. In the best mode fsr conducting the 22 invention, suah a molecule is obtained by cleaving a closed 23 covalent circular molecule to form a linear molecule.
24 Preferably, a restriction endonuclease capable of cleaving the molecule at a single site to produce either a blunt end 26 or staggered end linear molecule is employed. ~ost 27 preferably, the nucleotides on each side of this restriction 28 site will comprise at least a portion of the preferred two 29 regions of homology betw~en the DNA molecule being introduced and the cellular genome.
31 The invention thus provides a method for introduciny the 32 "desired gene sequence" into the genome of an animal or 33 plant at a specific chromosomal location. The "desired gene . .:

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WO91/19796 PCT/~'S91/04006 2r..~
l seque~ce" may be of any length, and have an~ nucleotide 2 sequence. It may comprise one or more qene sequences which 3 encode complete proteins, fragments of such gene sequences, 4 regulatory sequences, etc. Significantly, the desired gene sequence may differ only slightly from a native yene of the 6 recipient cell (for example, it may contain single, or multiple base alterations, insertions or deletions relative 8 to the native gene). The use of such desired gene sequences 9 will permit one to create subtle and precise changes in the genome of the recipient cell. Thus, the present invention ll provides a means for manipulating and modulatinq gene 12 expression and regulation. ;
13 In particular, the invention provides a mean for 14 manipulating and modulating gene expression and protein structure through the replacement of a gene sequence with a 16 "non-selectable" "desired gene sequence." A gene sequence 17 is ncn-selectable if its presence or expression in a -;
18 recipient cell provides no survival advantage to the cell l9 under the culturing conditions employed. Thus, by definition, one cannot select for cells which have received 21 a "non-selectable" gene sequence. In contrast, a "dominant"
22 gene sequence is one which can under certain circumstances 23 provide a survival advantage to a recipient cell. The 2~ neomycin resistance conferred by the n~tII gene is a survival advantage to a cell cultured in the presence of 26 neomycin or G418. The n~tII gene is thus a dominant, rather 27 than a non-selectable gene sequence.
28 In particular, the invention permits the replacement of 29 a gene sequence which is present in the recipient cell with an "analog" sequence. A sequence is said to ~e an analog o~
3l another sequence if the two sequences are su~stantially 32 similar in sequence, but have minor changes in sequence ~;-33 corresponding to single base substitutions, deletions, or ; ' 1' . . ~ , :... .: .
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1 insertions with respect to one anothe-, or if they possess 2 "minor" multiple ~ase alt~rations. Such alterations are 3 intended to exclude insertions of dominant selectable marker 4 genes.
When the desired gene sequence, flanked by regions of 6 homology with the recipient cell, is introduced into the 7 r~cipient cell as a linear double stranded molecule, whose 8 termini correspond to the regions of homology, a single 9 recombination event with the cell's genome will occur in 10 approximately 5% of the transfected cells. Such a single 11 recombinational event will lead to the integration of the 12 entire linear molecule into the genome of the recipient 13 cell.

14 The structure generated by the integration of the lineàr 15 molecule will undergo a subsequent, second recombinational 16 event (approximately 105 - 10-7 per cell generation). This 17 second recombinational event will result in the elimination 18 of all DNA except for the flanking regions of homology, and 19 the desired DNA sequence from the integrated structure.
Thus, the consequence of the second recombinational event 21 is to replace the DNA sequence which is normally present 22 between the flanking regions of homology in the cell's 23 genome, with the desired DNA sequence, and to eliminate the 24 instability of gene replacement.
The DNA molecule containing the desired gene sequence 26 may be introduced into the pluripotent cell by any method 27 which will permit the in~roduced molecule to undergo 28 recombination at its regions of homology. Some methods, 29 suc~ as direct microinjection, or calcium phosphate 30 transformation, may cause the introduced molecule ts form 31 concatemers upon integration. These concatemers may resolve 32 themselves to form non-concatemeric integration structures.
33 Since the presence of concatemers is not desired, methods : . . ,.................... .. .. . ~ . . :
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WO91/1~79~ PCT/~'S91/04006 1 which produce them are no~ preferred. In a preferred 2 embodiment, the DNA is introduced by electroporation 3 (Toneguzzo, F. et al., Nucleic Acids Res. 16:5515-5532 4 (1988); Quillet, A. et al., J. Immunol. 141:17-20 (1988);
Machy, P. et al., Proc. Natl. Acad. Sci. (U.S.A.) 85:8027-6 8031 (1988); all of whic~ references are incorporated herein 7 by reference). -8 After permitting the introduction of the DNA
9 molecule(s), the cells are cultured under conventional conditions, as are known in the art. -~
ll In order to facilitate the recovPry of those cells which 12 have received the DNA molecule containing the desired g~ne 13 sequence, it is preferable to introduce the DNA containing 14 the desired gene sequence in combination with a second gene ~-sequence which would contain a detectable marker gene 16 sequence. For the purposes of the present invention, any 17 gene sequence whose presence in a cell permits one to ~;
18 recognize and clonally isolate the cell may be employed as l9 a detectable marker gene sequenc:e.
In one embodiment, the presence of the detectable marker 21 sequence in a recipient cell is recognized by hybridization, 22 by detection of radiolabelled nucleotides, or by other 23 assays of detection which ~o not require the expression of 24 ~he detectable marker sequence. Preferably, such sequences are detected using PCR (Mullis, K. et al., Cold S~rinq 26 Harbor SvmP. Ouant. Biol. 51:263-273 (1986); Erlich H. et 27 al., EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, 28 K., EP 201,18~; Mullis X. et al., US 4,683,202; Erlich, 29 ~5 4,582,788; and Saiki, R. et al., US 4,683,194), which references are incorporated ~erein by reference).
31 PCR achieves the amplification of a specific nucleic 32 acid sequence using two oligonucleotide primers 33 complementary to regions of the sequence to be amplified.

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WO91/1979~ PCT/~'S91/04006 1 Extensio~ products incorporating the primers then become 2 templates for subsequent repllcation steps. PCR provides a 3 method for selectively increasing the concentration of a 4 nucleic acid molecule having a particular sequence ev~n when that molecule has not bee~ previously purified and is 6 present only in a single copy in a particular sample. The 7 method can be used to amplify either single or double 8 stranded DNA.
9 Most preferably, however, the detectable marker gene sequence will be expressed in the recipient cell, and will 11 result in a selectable phenotype. Examples of ~uch 12 preferred detectable gene sequences include the h~rt gene 13 (Littlefield, J.W., Science 145:709-710 (1964), herein 14 incorporated by reference), a xanthine-guanine phosphoribosyltransferase (q~t) gene, or an adenosine 16 phosphoribosyltransferase (aPrt) ge~e (Sambrook et_al., In:
17 Molecular Cl onlnq A LaboratorY Manual, 2nd. Ed., Cold Spring 18 Harbor Laboratory Press, NY (1989), herein incorporated by 19 reference), a tk gene (i.e. thymidine kinase gene) and especially the tk gene of herpes simplex virus (Giphart-21 Gassl2r, M. et al., Mutat. Res 214:223-232 (1989) herein 22 incorporated by reference), the nPtII gene (Thomas, K.R. et 23 al., Cell 51:503-512 (198~); Mansour, S.L. et al., Nature 24 336:348-352 (1988), both references herein incorporated by reference), or other genes which confer resistance to amino 26 acid or nucleoside analogues, or antibiotics, etc. Examples 27 o~ such genes include gene sequences which encode enzymes 28 such as dihydrofolate reductase (DHFR) enzyme, adenosine 29 deaminase ~ADA), asparagine synthetase (AS), hygromycin B
phosphotransferase, or a CAD enzyme (carbamyl phosphate 31 synthetase, asp rtate transcarbamylase, and dihydroorotase) 32 (Sambrook et al., In: Molecular Cloninq A Laboratorv Manual, : . , , . :- . . : ~ :
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WO91/l9796 PCT/~S91/04006 -31~

1 2nd. Ed., Cold Spring Harbor Laboratory Press, NY (1989), 2 herein incorporated by reference).
3 Cells that do not contain an active thymidine kinase 4 (TK) enzyme, a hypoxanthine-phophoribosyltransferase (HPRT) enzyme, a xanthine-guanine phosphoribosyltransferase (XGPRT) 6 enzyme, or an adenosine phosphoribosyltransferase (APRT) 7 enzyme, are unable to grow in medium containing 8 hypoxanthine, aminopterin, and/or mycophenolic acid (and 9 preferably adenine, xanthine, and/or thymidine), and thymidine, but are able to grow in medium containing 11 nucleoside analogs such as 5-bromodeoxyuridine, 6~
12 thioguanine, 8-azapurine, etc. (Littlefield, J.W., Science 13 145:709-710 (1964); Sambrook et al., In: Molecular Clonina 14 A Laboratorv Manual, 2nd. Ed., Cold Spring Harbor Laboratory Press, NY (1989)).
16 Conversely, cells that do con~ain such active enzymes 17 are able to grow in such medium, but are unable to grow in 18 medium containing nucleoside analogs such as 5-19 bromodeoxyuridine, 6-thioguanine, 8-azapurine, etc.
(Sambrook et al., In: Molecular Cloninq A Laporatorv Manual, 21 2nd. Ed., Cold Spring Harbor Laboratory Press, NY (1989)).
22 Cells expressing active thymidine kinase are able to 23 grow in media containing HATG, but are unable to grow in 24 media containing nucleoside analogues such as 5-azacytidine (Giphart-Gassler, M. et al., Mutat. Bes. 214:223-232 26 (1989)). Cells containing an active HSV-tk gene are 27 incapable of growing in the presence of gangcylovir or 28 similar agents.
29 The detectable marker gene may be any gene which càn complement for a racognizable cellular deficiency. Thus, 31 for example, the gene for HPRT could be use~ as the 32 detectable marker gene sequence when employing cells lacking 33 HPRT activity. Thus, this gene is an example of a gene .
.

.

w o 91/19796 PCT/~S91/04006 1 whose expression product may be used to select mutant cells, 2 or to "negatively select" for cells which express this gene 3 product.
4 The n~tII gene (Southern, P.J., et al., J. Molec. A~l.
S Genet. 1:327-341 (1982); Smithies, O. et al., ~ature 6 317:230-234 (1985), which references are incorporated herein 7 by reference) is the most preferred detectable marker gene 8 sequence. Constructs whi~h contain both an nPtII gene and 9 either a tk gen~ or an hprt gene are especially preferred.

11 A. ~ e of a ~ingle DNA ~olecule cont~i~ing Both the 12 Detectable M~r~r 8equence and the Desired Gene 13 ~eque~ce 1~ In a first preferred embodiment, the detectable marker 16 gene sequence, flanked by- the regions of homology, is 17 provided to the recipient cells on the same DNA molecule 18 which contains the desired genP sequence. As discussed 19 previously, it is preferred that this DNA molecule be a linear molecule.
21 After selection for cells which have incorporated the 22 desired DNA molecule (for example by selection for G418 23 resistant cells when the detectable marker gene sequence is 24 an expressible nptII gene sequence), the cells are cultured, and the presence of the introduced DNA molecule is confirmed 26 as described above. Approximately 107 cells are cultured and 27 screened for cells which have undergone the second 28 reco~binational event (discussed above) resulting in the 29 replacement of a native se~uence (i.e. a gene sequence which is normally and naturally present in the recipient 31 cell) with the desired gene se~uence.
32 Any of a variety of methods may be used to identify 33 cells which have undergone the second recombinational event.

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wvsl/19796 PCrt~'S9l/04006 ~.,1`, , ~ F 7 1 1 Direct screening of clones, use of PcR, use of hybridization 2 probes, etc., may all be employed for this purpose. In a 3 preferred embodiment, the DNA molecule will, in addition to 4 the desired gene sequence, the flanking regions of homology and the detectable marker gene sequence, contain an 6 additional gene sequence which will permit the selection or 7 recognition of cells which have undergone the second 8 recombinational event. This additional gene sequence will 9 be excised from the cell's genome as a direct consequence of the second recombinational event. Thus, gene sequences 11 which are sui~able for this purpose include any gene 1~ sequence whose loss from a cell can be detected or selected 13 for. Examples of such "negative selection" gene sequences 14 include the hPrt gene, and the tk gene (especially the tk gene of herpes simplex virus).
16 In the first preferred embodiment, the frequency of the 17 second reco~binational event is approximately 105. However, 18 the use of a "negative selection" gene sequence permits one 19 to identify such recombinant cells at a frequency of approximately 100%.
21 As illustrated in Figure 2, the DNA molecule may have a 22 region of heterology located at the proposed insertion site.
23 Insertion of such a vector permits one to select for 24 recombinants which have recombined at the insertion site (and not at other potential sites). If recombina~ion occurs 26 at the desired insertion site, it will lead to the loss of 27 the sequence o~ heterology located at the proposed insertion 28 site o~ the DNA molecule (~SVtk, for exam~le, in Figure 2A).
29 Insertions which result from other recombinational events will retain the sequence of heterology. Thus, by e~ploying 31 a region of heterology which encodes ~n assayable gene 32 product, or which can be used as a "negative selectable"
33 =arker, one oan readily determine thet the loous of '~

-WO 91/19796 P~ 'S91/04006 .
~ t~ 34-l insertlon of the recipient cell contains the precise 2 sequence desired. As indicated in Figure 2A), the 3 efficiency of such a vector is approximately 1/197.
4 The region of heterology which may be introduced at the insertion site of t~e DNA molecule may be either short tfor 6 example, 26 base pairs, Figure 2B) or of substantial size 7 (or example, 2 kb, Figure 2A). The site of linearization 8 may be 5', 3', or within the region of heterology. When the 9 site of linearization is within the region of heterology, the efficiency of gene targeting is l/63.
11 As shown in Figure 2c, the region of heterology may be 12 located at a site internal to the region of homology where 13 the desired recombination shall occur. Such a construct can 14 be used when one desires to introduce a subtle mutation into i5 a locus of the cellular gene at a site other than that of 16 the site of desired recombination~

18 B. n3e of a Diff~re~t DNA ~olecules to Pro~i~e the 19 DetectAble Marker 8egue~e and t~e Desired Gene 8equence 22 In a second preferred embodiI~ent, the detectable marker 23 gene sequence, flanked by the regions of homology, will be 24 provided to the recipient cell on a different DNA molecule from that which contains the desired gene sequence. It is 26 preferred that these molecules be linear molecules.
27 When provided on separate DNA molecules, the detectable ~8 marker gene sequence and the desired gene sequence will most 29 preferably be provided to the recipient cell by co~
electroporation, or by other Pqui~alent techniques.
31 After selection of such recipients (preferably through 32 the use of a detectable marker sequence which expresses the 33 nptII gene and thus confers cellular resistance to the . ., .: :, : . . : : - :- .

, ' ' ' `~ :

WO91/~9796 PCT/~S91/04006 -35~ ~7~ ~

1 antibiotic G418), the cells are gro~n up and screened to 2 confirm the insertion event (preferably using PCR).
3 In the absence of any selection, only one cell in 107 4 would be expected to have the predicted recombinant structures. If, however, one selects for recipient cells 6 which contain and express a detectable marker sequence (such 7 as the nPtII gene), it is possible to obtain a 103 to 105 8 fold enrichment for cells which have taken up both DNA
9 molecules. Typically, such enrichment enables one to identify the desired recipient cell (in which the introduced 11 DNA has in~egrated into the cell's genome) by screening only 12 800 -1,500 cells. Such screening is preferably done using 13 PCR, or other equivalent methods. Using such negative 14 selection techniques, one may manipulate the vector copy number.
16 The two introduced DNA molecules will generally not have 17 integrated into the same site in the genome of the recipient 18 cell. Thus, in some cases, the desired gene sequence will 19 have integrated in a manner so as to replace the native cellular gene sequence between the flanking regions of 21 homology. The locus of integration of the detectable marker 22 gene is unimportant for the purposes of the present 23 invention, provided it is not genetically linked to the same 24 locus as the desired gene sequence. If desired, however, it is possible to incorporate a gene sequence capable of 26 negative selection along with the DNA containing the 27 detectable marker sequence. Thus, one can ultimately select 28 for cells which have lost the introduced selectable marker 29 gene sequence DNA.

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~ r ~ 3 6--1C. ~se of Direct Bel~ction to I~entify ~omolog~us 2Recombin~tion Event~

4Although all of the above-described preferred 5embodiments enable the isolation of cells in which one of a 6cell's alleles has been mutated to contain a desired gene 7sequence, each embodiment requires the screening of a 8significant number of candidate cells in order to identify 9the desired recombinant cell. It is, however, possible to 10directly select for the desired recombinant cell by 11employing a variation of the above embodiments. This 12embodiment of the invention is illustrated in Figure 13. In 13the methods illustrated in Figure 13, if the sequence 14located below the asterisk is a neo gene, then only tha 15mutant revertants will be selected if 6-thioguanine and G418 16selection is applied to select for the excision events.
17The method for direct selection of the desired cells 18relies upon the phenotypic difference in targeted and non-19targeted cells and the use of a single gene which can be 20used for both positive and negative selection.
21Typically, in any homologous recombination experiment 22performed with an insertion vector, three populations o~
23cells will be created. The first class of cells will be 24those which have failed to receive the desired DNA molecule.
25This class will comprise virtually all of the candidate 26cells isolated on completion of the experiment. The second 27cl~ss of cells will be those cells in which the desired gene 28seguence has been incorporated at a random insertion site 29(i.e. a site other than in the gene desired to be mutated).

30Approximately one cell in 103-10~ total cells will be in this 31class. The third class of cells will be those cells in 32which the desired gene sequence has been incorporated by 33homologous recombination into a site in ~he desired gene.

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wo91/l9796 PCT/~S91/04006 g ~

1 Approximately one cell in l05-106 total cells will be in thls 2 class.
3 In the above-descrlbed embodiments, the cells of the 4 first class (non-transfected cells) can be eliminated by positive selection, thus necessitating the scxe~ening of only 6 about 1,000 cells in order to identify the desired 7 recombinant oell. In the present embodiment, cells of the 8 third class (homologous recombinants) may be selected from 9 the cells of the second class (random insertions) if a phenotypic difference exists between the cells of the two 11 classes.
12 Since random integration sites are likely to be 13 concatemeric with few single copy clones (depending upon the 14 DNA concentration with which the cells were transfected?, such inteyration events are inherently unstable. Thus, such 16 concatemeric constructs will typically undergo intrachromo-17 somal recombination. Such reco~bination will always leave 18 one intact copy of the vector in the genome. Thus, all 19 random insertion events may be negatively selec~ed from the population if a negatively selectable marker is included on 21 the vector.
22 In contrast, cells in which the desired gene sequence 23 has been incorporated into the desired gene by homologous 24 recombination will revert with a relatively high frequency ~approximately 1 in 104-105 per cell division (depending upon 26 the size of the duplicated structure) to produce a mutated 27 desired gene that does not contain vector sequences.
28 There~ore, even if the vector contained a negatively 29 selectable gene sequence, such cells will survive negative selection, and can be recovered. The majority o~ ho~ologous 31 reco~binant cells do not undergo reversion, and will be 32 eliminated by the negative selection. Thus, the sum of th~

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WO 91/19796 PCI/~591/04006 1 selections will result in the isolation of the desired 2 recombinants.
3 The method comprises incubating a "precursor cell" (i.e.
4 a cell which is to be changed by application of the method S into the "desired" recombinant cell) under non-selective 6 culture conditions, or under a first s~t of selective 7 culture conditions. A culturing condition (i.e. medium, 8 temperature, etc.) is said to be "non-selective" if it is 9 capable of promoting the growth (or sustaining the viability) of a precursor cell, a desired cell, and an 11 intermediate cell type ti.e. a cell obtained during the 12 progression of a precursor cell into a desired cell). A
13 culturing condition is said to be "selective" if it is 14 capable of promoting the growth (or sustaining the viability) of only certain cells (i.e. those having a 16 particular genotype and which therefore contain a particular 17 gene product in either an active or an inactive form)~
18 Preferred selective culturing conditions thus depend l9 upon the genotype of the precursor cell. As stated above, cells that do not contain an active thymidine kinase (TX) 21 enzyme, a hypoxanthine-phophoribosyltransferase (HPRT) 22 enzyme, a xanthine-guanine phosphoribosyltransferase (XGPR~) 23 enzyme, or an adenosine phosphoribosyltransferase (APRT) 24 enzyme, are unable to grow in medium containing hypoxanthine, aminopterin, and/or mycophenolic acid (and 26 preferably adenine, xanthine, and/or thymidine), and 27 thymidine, but are able to grow in medium containing 28 nucleoside analogs such as 5-bromodeoxyuridine, 6-29 thioguanine, 8-azapurine, etc. Conversely, cells that do contain such active enzymes are able to grow in such medium, 31 but are unable to grow in medium containing nucleoside 32 analogs such as S-bromodeoxyuridine, 6-thioguanine, 8-33 azapurine, etc.
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WO 91/19796 PCr/ l 'S9 1 /O~l006 ~35~
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1 Such incubation is conducted in the presence of a DMA
2 molecule containing a desired non selectable gene sequence.
3 Preferably, the DNA molecule additionally contains two 4 regions of homology which flank this desired gene sequence, and which are sufficient to permit the desired gene sequence 6 to undergo homologous recombinatio~ with a predetermined 7 genP sequence of the genome of the precursor cell. The DNA
8 molecule additionally contains a selectable gene sequence 9 whose presence or expression in the cell can be selected for by culturing the cell under a first set of selective culture 11 conditions, and whose presence or expression in the cell can 12 be selected against ~y culturing the cell under a second set 13 of selective culture conditions.
14 Examples of preferred selectable gene sequences include gene sequences which encode an active thymidine kinase (TK) 16 enzyme, a hypoxanthine-phophoribosyltransferase (HPRT) 17 enzyme, a xanthine-guanine phosphoribosyltransferase (XGPRT) 18 enzyme, or an adenosine phosphoribosyltransferase (APRT) 19 enzyme. Such gene sequences can be used for both positive and negative selection.
21 Additional gene sequences which can be used as 22 selectable gene sequences include those which encode enzymes 23 such as dihydrofolate reductase (DHFR) enzyme, adenosine 24 deaminase tADA), asparagine synthetase (AS), hygromycin B
phosphotransferase, or a CAD enzyme (carbamyl phosphate 26 synthetase, aspartate transcarbamylase, and dihydroorotase).
27 Methods for producing cells deficient in expressing these 28 enzymes are described by Sambrook et al. (In: Molecular 29 Cloninq_~ LaboratorY Manual, 2nd. Ed., Cold Spring ~arbor Laboratory Press, NY (1989), herein incorporated by 31 reference). Such gene sequences can be used only for 32 positive selection.

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-4c-l The incubation is performed under conditions sufficient 2 to permit the DNA molecule to be introduc~d into the 3 precursor cell. Such introduced DNA molecules are able to 4 then undergo homologous reco~bination with the predetermined gene sequence of the genome of the precursor cell to thereby 6 produce the desired cell wherein the desired non-selectable 7 gene sequence has been inserted into the predetermined gene 8 sequence.
9 Such a desired cell can be recovered by culturing the cell under the first set of selective culture conditions, by l1 then permitting the cell to undergo intrachromosomal 12 recombination under non-selective culture conditions, and by-13 then incubating the cell under the second set of selective 14 culture conditions.
Thus, in one preferred embodiment, the precursor cell 16 lacks an active hypoxanthine-phophoribosyltransferase (HPRT) 17 enzyme, a xanthine-guanine phosphoribosyltransferase (XGPRT) 18 enzyme, or an adenosine phosphoribosyltransferase (APRT) 13 enzyme, and the selectable gene sequence expresses an active HPRT, XGPRT or APRT enzyme. In 1:he first set of selectable 21 culture conditions, medium containing hypoxanthine, 22 aminopterin and/or mycophenolic acid (and preferably 23 adenine, xanthine, and/or thymicline) is employed. In the 24 second set of selectable cult:uring conditions, medium containing a nucleoside analog such as 5-bromodeoxyuridine, 26 6-thioguanine, 8-azapurine, etc., is employed.
27 In a second preferred embodiment, the precursor cell 28 lacks an active TK enzyme, and the selectable gene sequence 29 expresses an active TK enzyme. In the first et of selectable culture conditions, medium containing 31 hypoxanthine, aminopterin, and thymidine is employed. In 32 the second set of selectable culturing conditions, medium 33 containing a thymidine analog such as FIAU (Borrelli, Proc.

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WO91/197g6 PCT/~S91/0~00~

rr~.`7~l 1 Nat'l. Acad. Sci. (U.S ~.) 8~:7s72 (1988), or gangcyclovir, 2 etc. is employed (if an HSV t~. gene is used), or 5-3 bromodeoxyuridine, etc. ( lf a cellular tk gene is employed).
4 A preferred negative selectable marker is the hprt gene (cells expressing an active HPRT enzyme are unable to gro~
6 in the presence of certain nucleoside analogues such as 6-7 thioguanine, etc.). When using S-~hioguanine as ~ negative 8 selection agent, a density of 104 cells / cm2 is preferably 9 used since the efficiency of 6-thioguanine selection is cell density dependent. A typical experiment with 107transfected 11 cells would yield approximately 10 revertant cells after - -12 successive selection. The relative yield of revertant 13 clones can be substantially increased by using "Poly A -~
14 Selection" for the first round of selection. "Poly A ~
Selection" is discussed in detail in Exa~ple 6 below. - -17 IV. The Pro~uction of Chi~eric and Tr~nsgenic Animsl~

l9 The chimeric or transgenic: animals of the present -invention are prepar~d by int:roducing one or more DNA
21 molecules into a precursor pluripotent cell, most preferably 22 an ES cell, or equivalent (Ro~)ertson, E.J., In: Current 23 Communica~ions in Molecular Bio]oay, Capecchi, M.R. (ed.), 24 Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), pp.
39-44, which reference is incorporated herein by reference).
26 The term ~'precursor" is intended to denote only that the 27 pluripotent cell is a precursor to the desired 28 ("transfected"~ pluripotent cell which i~ prepared in 29 accordance with the teachings of the present invention. The pluripotent (precursor or transfected) cell may be cultured 31 in VlVo, in a manner known in the art (Evans, ~.J. t al., 32 ~ature 292:154-156 (19 1)) to form a chi~eric or transgenic 33 ani~al.

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~ -r r f '- --~ 2--1 Any Es cell may be used ln accordance with the present 2 inve~tion. It is, however, preferred to use primary 3 isolates of ES cells. Such isolates may be obtained 4 directly from embryos such as the CCE cell line disclosed by Robertson, E.J., In: Current Communications _ln Molecular 6 Bioloqv, Capecchi, M.R. (ed.), Cold Spring Harbor Press, 7 Cold Spring Harbor, NY (1989), pp. 39-44), or from the 8 clonal isolation of ES cells from the CCE cell line 9 (Schwartzberg, P.A. et al., Science 246:799-803 (1989), which reference is incorporated herein by reference). Such 11 clonal isolation may be accomplished according to the method 12 of E.J. Robertson (In: Teratocarcinomas and Embrvonic Stem 13 Cells: A Practical ADProach, (E.J. Robertson, Ed.), IRL
14 Press, Oxford, 1987) which reference and method are incorporated herein by reference. The purpose of such 16 clonal propagation is to obtain ES cells which have a 17 greater efficiency Eor differentiating into an animal.
18 Clonally selected ES cells are ~pproximately 10-fold moxe l9 effective in producing transgenic animals than the progenitor cell line CCE. For the purposes of the 21 recombination methods of the present invention, clonal 22 selection provides no advantage. An example of ES cell 23 lines which have been clonally derived from embryos are the 24 ES cell lines, ABl (h~rt~) or AB2.1 (hprt).
The ES cells are preferably cultured on stomal cells 26 (such as STO cells (especially SNC4 STO cells) and/or 27 primary embryonic fibroblast cells) as described by E.J.
28 Robertson (In: Teratocarcinomas and Embrvonic Stem Cells: A
29 Practical Ap~roach, (E.J. Robertson, Ed.), IRL Press, Oxford, 1987, pp 71-112), which reference is incorporated 31 herein by reference. The stomal (and/or fi~roblastj cells 32 serve to eliminate the clonal overgrowth of abnormal ES
33 cells. Most preferably, the cells are cultured in the :~' WO 91/19796 PCI`/--IS91/04006 -43- ~ q~i~

1 presence of leukocyte inhibitory factor ("lif") (Gough, N.M.
2 et al., Reprod. Fertil. Dev. 1:281-288 (1989); Yamamori, Y.
3 et al., Science 246:1412-1416 (1989), both of which 4 references are incorporated herein by reference). Since the gene encoding lif has been cloned (Gough, N.M. et al., 6 ReProd. Fertil. Dev. 1:281-288 (1989)), it is especially 7 preferred to transform stomal cells with this gene, by means 8 known in the art, and to then culture the ES cells on 9 transformed stomal cells that secrete lif into the culture medium. -ll ES cell lines may be derived or isolated from any 12 species (for example, chicken, etc.), although cells derived 13 or isolated from mammals such as rodents (i.e. mouse, rat, 14 hamster, etc.), rabbits, sheep, goats, fish, pigs, cattl~, primates and humans are preferred.

17 V. The Production of ~hi~eric a~d Tr~nsgenic Plant~

19 The chimeric or transgenic plants of the invention are produced through the regeneration of a plant cell which has 21 received a DNA molecule through the use of the methods 22 disclosed herein.
23 All pla~ts from which protc~plasts can be isolated and 24 cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered 26 which contain the introduced gene sequence. Some suitable 27 plants include, for example, species from the genera 28 Fra~aria, Lotus, Medic~qo, OnobrYchis, Trifolium, 29 Triaonella, Viana, Citrus, Linum, Geranium, Manicot, Daucus, Arabido~sis, Brassica, Ra~hanus, Sina~is, Atro~a, Cavsicum, 31 Datura, Hyosc~amus, Lvco~ersion, Nisotiana, Solanum, 32 Petunia, Pi~italis, Maiorana, Cich rium, Helianthus, 33 Lactuca, Bromus, AsParaaus, Antirrhinum, Hemer-callis, ,.................. ~ .. , . . : , . . .
.. .. ...

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~44--1 Nemesia, Pelaraoniur~, Panicum, Pennisetur~, Ranlln~ulus, 2 Senecio, Salpiqlossis, Cucumis, Browallia, Glycine, Lollum, 3 Zea, Triticum, Sorqhum, Ipomoea, Passiflora, CYclamen, 4 Malus, Prunus, Rosa, Rubus, Populus, Santalum, Allium, Lilium, Narcissus, Ananas, Ara~his, Phaseolus, Pisum and 6 Datura.
7 There is an increasing body of evidence that practically 8 all plants can be regenerated from cultured cells or 9 tissues, including but not limited to all major cereal crop species, sugarcane, sugar beet, cot~on, fruit and other 11 trees, legumes and vegetables.
12 Plant regeneration from cultural protoplasts is 13 described in Evans et al., "Protoplast Isolation and 14 Culture," in Handbook of Plant Cell Culture 1:124-176 (MacMillan Publishing Co., New York, 1983); M.R. Davey, 16 "Recent Developments in the Culture and Regeneration of 17 Plant Protoplasts," Proto~lasts, 1983 - Lecture Proceedings, 18 pp. 19-29 (Birkhauser, Basel, 1983); P.J. Dale, "Protoplast 19 Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops," in P to~lasts 1983 - Lecture 21 Proceedings, pp. 31-41 (Birkhauser, Basel, 1983); and H.
22 Binding, "Regeneration of Plants," in Plant Proto~lasts, pp.
23 21-37 (CRC Press, Boca Raton, 1985).
24 Regeneration varies from sp~ecies to species of plants, but generally a suspension of transformed protoplasts 26 containing the introduced gene sequence is formed. ~mbryo 27 formation can then be induced from the protoplast 28 ~uspensions, to the stage of ripening and germination as 29 natural embryosO ThP culture media will generally contain various amino acids and hormones, such as auxin and 31 ~ytokinins. It is also advantageous to add glutamic acid -`~
32 and proline to the medium, especially for such species as 33 corn and alfalfa. Shoots and roots normally develop :

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1 simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the histor~ of the culture.
3 If these three variables ar~ controlled, then regeneration q is fully reproducible and repeatable.
The mature plants, grown from the transformed plant 6 cells, are selfed to produce an inbred plant. The inbred 7 plant produces seed containing the introduced gene sequence.
8 These seeds can be grown to produce plants that express this 9 desired gene sequence. ~
Parts obtained from the regenerated plant, such as ~ -11 flowers, seeds, leaves, branches, fruit, and the like are 12 covered by the invent1on. Progeny and variants, and mutants 13 of the regenerated plants are also included within the scope 14 of this invention.
As used herein, variant describes phenotypic changes 16 that are stable and heritable, including heritable variation 17 that is sexually transmitted to progeny of plants.
18 ~ -19 VI . GENE E~PRESSION

21 In one embodiment, the DNA molecule(s) which are to be 22 introduced into the recipient cells in accordance with the 23 methods of the present inventioIl will be incorporated into 24 a plasmid or viral vector (or a derivative thereof) capable of autonomous replication in a host cell.
26 Preferred prokaryotic ~ectors include plasmid6 such as 27 those capable of replication in E. coli such as, for 28 example, pBR322, ColEl, pSC101, pACYC 184, ~X. Such 29 plasmids are, for example, disclosed by Maniatis, T., et al. ~-tIn Molecular Clonina. A Laboratorv Manual, Cold Spring 31 Harbor Press, Cold Spring Harbor, NY (1982)). Bacillus 32 plasmids include pClg4, pC221, pT127, etc. Such plasmids ~;~
33 are disclosed by Gryczan, T. (In: The Molecular Biolo~v of ;

... . . .. . . . . .

W O 91/19796 P ~ /~S91/04006 1 the ~acilli, Academic Press, NY (1982), pp. 307-329).
2 Suitable StrePtomvces plasmids include pIJ101 (Kendall, 3 K.J., et al., J. Bacteriol. 169:4177-4183 (1987~), and 4 Stre~tomyces bacteriophages such as ~C31 (Chater, K.F., et al., In: Sixth International S~m~osium on Actinomvcetales 6 Bioloqv, AXademiai Xaido, Budapest, Hungary (1986), pp. 45-7 54). Pseudomonas plasmids are reviewed by John, J.F., et 8 al. (Rev. Infect. Dis. 8:693-704 (1986)), and Izaki, K.
9 (Jpn. J. Bacteriol. 33:729-742 (1978)).
Examples of suitable yeast vectors include the yeast 2-11 micron circle, the expressi~n plasm~ds YE~13, YCP and YRP, 12 etc., or their derivatives. Such plasmids are well known in 13 the art (Botstein, D., et al., Miami wntr. S~mp. 19:265-274 }4 (1982); Broach, J.R., In: The Molecular Bioloqv of the Yeast Saccharomy~es: Life Cvcle and Inheritance, Cold 16 Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 17 (1981); Broach, J.R., Cell 28i~i203-204 (1382)).
18 Examples of vectors which may be used to replicate the 19 ~NA molecules in a mammalian host include arlimal viruses such as bovine papill~ma viruci, polyoma virus, adenovirus, 21 or SV40 virus.

23 VII. ~e3 of the PraYent Inventio~

The methods of the present invention permit the 26 introduction of a desired ~ene sequence into an animal or 27 plant cell.
28 In a first embodiment, the methods of the present 29 invention may be uied to introduce DNA into germ line cells of animals in order to produce chimeric or transgenic 31 animals which contain a desired gene sequence. The animals 32 which may be produced through application of the described 33 method include chicken, non-human mammals (especially, . , . ' .. ' . .

WO9l/19~96 PCT/~'S9lJ0~006 -~7-1 rodents (i.e. mouse, rat, hamster, etc.), rabbits, sheep, 2 goats, fish, pigs, cattle and non-human primates).
3 As stated above, the desired gene sequence may be of any 4 length, and have any nucleotide sequence. In particular, it is possible to design the sequence of the desired gene 6 sequence in order to create single, or multiple base 7 alterations, insertions or deletions in any preselected gene 8 of a cell.
9 If such changes are within a translated region of a native gene sequence, then a nPw protein variant of a native 11 protein can be obtained. Such a procedure can, for example 12 be used to produce animals which produce improved (i.e. more 13 stable, more active, etc.) enzymes, binding proteins, 14 receptors, receptor ligands, etc.
The methods of the present invention may be used to 16 produce cells in which a natural gene has been replaced with 17 a heterologous gene. A gene is said to be heterologous to 7 8 a transgenic cell if it is derivable from a species other 19 than that of the transgenic cell.
In one embodiment, this replacement may be accomplished 21 in a single step (Figure 3). To accomplish such 22 replacement, a DNA molecule containing a desired gene 23 sequence and a region of homology is introduced into a 24 recipient cell. A selectable maeker gene is also introduced into the cell, and used to select for cells which have 26 underwent recombination. The method results in the -27 replacement of the normal sequences ad~acent to the region 28 of homology with the heterologous sequences of the desired 29 DNA sequence.
3~ In a second embodiment, this replacement may be 31 accomplished in a two steps (Yigure 4). A~ in the 32 embodiment described above, a cell is provided with a DNA
33 molecule containing a desired gene sequence and a r~gion of - .
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WO 91/~9796 PCr/-S91/0400S

1 homology. The DNA molecule also contains a selectable 2 marker gene used to select for cells which have undergone a 3 recombinational event that has resulted in the insertion of 4 the introduced DNA molecule into their chromosomes at the site of homology. The structure of such an insertion site 6 is depicted in Figure 4A.
7 Significantly, in this embodiment, the introduced DNA
8 ~olecule will al50 contain a "negative selectable" marker 9 gene which can be used to select for cells which undergo a second recombinational event that results in the loss of the 11 inserted DNA.
12 As shown in Figure 4B, a second DNA molecule is employed 13 to complete the gene replacement. This second DNA molecule 14 need not contain any selectable marker gene. ~pon receipt of the second DNA molecule, a second recombinational event 16 occurs which exchanges the "second" DN~ molecule for the 17 integrated "first" DNA molecule (including the desired DNA
1~ sequence, the selectable marker sequence, and the "negative 19 selectable" marker sequence contained on that molecule).
This aspect of the invention is illustrated in Figure 4B.
21 In another embodiment of the invention, subtle mutations 22 may be introduced into a desired locus using a "cassette"
23 construct containing both a pos:itive selection marker (such 24 as the n~tII gene or the qpt gene~ and a negative selection marker (such as the tk gene). In this embodiment, one first 26 uses the positive selection capacity of the construct to 27 introduce the two selection markers into a desired ~ocus.
28 One then introduces the desired subtle mutations (substi-29 tutions, insertions, deletions, etc.) by providing a cell with a DNA molecule that contains the desired ~utation. By 31 selecting for the loss of the "cassette" (using the negative 32 selection marker), one can select for recombinational events 33 which result in the replacement of the "cassette" sequence ~' .

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, wosl/l979~ PCT/~S91/04006 -49~

l with the DNA sequence containing the desired mutation. This 2 embodiment of the invention is illustrated in Figure 5.
3 The methods of the present invention may also be used tc 4 replace contiguous regions of a chromosome with any desired gene sequence. Thus, the present invention is not limited 6 in the size of the DNA regions which may ~e altered or 7 replaced. This aspec~ of the present invention is 8 illustrated in Figure 6, as a series of 5 steps (Figures 6A-9 6E). The method is applicable to any gene sequence. It is especially useful in producing cells which contain 11 heterologous immunoglobulins (such as the heavy chain locus 12 of an immunoglobulin).
13 The first step in replacing a large region of a 14 chromosome with a desired sequence involves setting up an initial target. In this step, a recipient cell is provided 16 with a DNA molecule which contains a "first fragment" of the 17 total desired replacement sequence (Figure 6A). This "first 18 ~ragment" of the desired replacement sequence contains a l9 selectable marker sequence (most preferably the nPtII gene) at its end.
21 The DNA molecule also contains a "dual selection" gene 22 sequence which encodes a non-functional fra~ment of a gene 23 sequence for which both a positive and a negative selection 24 exists. An example of such a gene is the oDt gene when used in the context of an h~rt- cell. Cells which express a 26 functional qpt gene can be selected for by their ability to 27 grow in HAT medium; Cells which lack a functional ~t gene 28 can be selected for by their ability to grow in the presence 29 of 6-thioguanine.
Homologous recombination results in the insertion of the 31 DNA molecule into the cell's genome at the region of 32 homology (Figure 6A). Importantly, since this step results 33 in the creation of a cell whose genome contains the .
; ~ : ., : .i ' , . .::
' ~ ' WO 91/19796 PCT/~'S91/04006 f ~ --50--1 selectable marker gene, it is possible to sel~ct for the 2 desired reco~binational event.
3 In the second step of the method, a second DNA molecule 4 is provided to the cell. This second DNA molecule contains a "second fragment" of the desired replacement sequence as 6 well as a sequence of the dual selection gene that, due to 7 an internal deletion, is incapable of encoding a functional 8 gene product. Homologous recombination results in the 9 insertion of the second DNA molecule into the cell's genome in a manner so as to create a functional dual selection gene 11 (Figure 6B). Recombination also results in the integration 12 of a non-functional fragment of the dual selection gene.
13 Importantly, since this step results in the creation of a 14 cell whose genome contains a functional dual selection gene, it is possible to select for the desired recombinational 16 event.
17 In the third step of the method, a third DNA molecule is 18 provided to the cell. This third DNA molecule contains both 19 the "first" and l'second" fragments of the desired replacement sequence. Homologous recombination results in 21 the insertion of the third DN~ molecule into the cell's 22 genome in a manner so as to delete the functional dual 23 selection gene. The non-functional fragment of the dual 24 selection gene (formed in step ~) is not affected by the recombination, and is retained (Figure 6C). Importantly, 26 since this step results in the creation of a cell whose 27 genome lacks the dual selection gene, it is possible to 28 select for t~e desired recombinational event.
29 In ~he fourth step of the method, a ~ourth DNA molecule is provided to the cell. This fourth DNA molecule contains 31 a "third fragment" of the desired replacement sequence as 32 well as a seguence of the dual selection gene that, as in 33 step 2, is incapable of encoding a functional gene product .:: . . . ~

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Wo 91/1979h PCl/~S91/04006 - 5 1 ~ ~' f~

l due to an internal deletion. Homologous recombinatio~
2 results in the insertion of the fourth DNA molecule into the 3 cell's genome in a manner so as to create a functional dual 4 selection gene (Figure 6D), Recombination also results in the integration of a non functional fragment of the dual 6 selection gene. Importantly, since this step results in the 7 creation of a cell whose genome contains a functional dual 8 selection gene, it is possible to select for the desired 9 recombinational event.
In the fifth step of the method, a fifth DNA molecule is 11 provided to the cell. This fifth DNA molecule contains both 12 the "second" and "third" fragments of the desired 13 replacement sequence. Homologous recombination results in 14 the insertion of the fifth DNA molecule into the cell's genome in a manner so as to delete the ~unctional dual 16 selection gene. The non-functional fragment of the dual 17 selection qene (formed in step 4) is not af~ected by the 18 recombination, and is retained (Figure 6C). Importantly, l9 since this stPp results in the creation of a cell whose genome lacks the dual selection gene, it is possible to 21 select for the desired recombinational event.
22 As will be appreciated, the net ef~ect of the above-23 described steps is to produce ,a cell whose genome has been 24 engineered to contain a "first," "second," and "third"
"fragment" of a particular desired gene in a contiguous 26 ~anner. The steps may be repeated as desired in order to 27 introduce additional "fragments" into the cell's genome. In 28 this manner, cells can be constructed which contain 29 heterologous genes, chromosome fragments, or chromosomes, that could not be introduced using a single vector. As 31 indicated above, each step of the method can be selected 32 for.

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: '' ' ~ -,:, ~ ,,. . , - ~ '' '"' . ~ ' 9l/19796 PCT/~591/04006 ~ 52--1 In particular, this aspect of the present invention may 2 be used to produce "humanized" antibodies (i.e. non-human 3 antibodies which are non-immunogenic in a human) (Robinson, 4 R.R. et al., Interna~ional Patent Publication PCT/US86/02269; AXira, K. et al., European Patent 6 Application 184,187; Taniguchi, M., European Patent 7 Application 171,4g6; Morrison, S.L. et al., European Patent 8 Application 173,494; Neuberger, M.S. et al., PCT Application 9 Wo 86/01533; Cabilly, S. et al., European Patent Application 125,023; Better, M. et al., science 240:1041-1043 (1988);
11 Liu, A.Y. et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 12 ~1987); Liu, A.Y. et al., J. Immunol. 139:3521-3526 (1987);
13 Sun, L.K. et al., Proc. Natl. Acad. Sci. USA 84:214-218 14 (1987~; Nishimura, Y. et_al., Canc. Res. 47:999-1005 (1987);
Wood, C.R. et al., Nature 314:446-449 (1985)); Shaw et al., 16 J. Natl.Cancer Inst. 80:1553-1559 (1988).
17 The method may also be used to produce animals having 18 superior resistance to disease, animals which constitute or 19 produce improved food sources, animals which provide fibers, hides, etc. having more desirable characteristics. The 2~ method may also be used to produce new animal models for 22 human genetic diseases. For example, the method may be used 2~ to "humanize" the CD4 analog of an animal, and thus provide 24 an animal model for AIDS. SUCh animal models can be used for drug testing, and thus hasten the development of new 26 therapies for genetic diseases.
27 In addition, the present invention permits the formation 28 of cells and of transgenic animals which contain mutations 29 in ~edically or clinically significant heterologous genes.
A gene is said to be medically or clinically significant if 31 it e~presses an isotype of a protein associated with a human 32 or animal disease or condition. Examples of such genes 33 include the genes which encode: topoisomerase pl80, 5~

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~: , ' ' '- . . . 1 Wo91/l9796 PCT/~S91/~4006 1 reductase, ACAT, 5-lipoxygenase, the insulin receptor, the 2 interleukin-2 receptor, the epidermal growth factor 3 re~eptor, the seratonin receptor, the dopamine receptor, the 4 GABA receptor, the V2 vasopressin receptors, G proteins (signal transduction), phospholipase C proteins, and 6 insulin. A transgenic mouse produced by microinjection 7 which expresses human insulin was reported by Selden, R.F.
8 et al. (European Patent Publication No. 247,494, which 9 reference is incorporated herein by reference).
The transgenic cells and animals discussed a~ove can be 11 used to study human gene regulation. For example, 12 transgenic animals which express a human isotype of 13 topoisomerase pl80, 5-~ reductase, ACAT,S-lipoxygenase, or 14 hormone or cytokine receptors would have ultility in in vivo drug screening. The expression of topoisomerase pl80 is 16 associated with resistance to chemotherapeutics. Thus, 17 agents which interfere with this enzyme could be used to 18 enhance the effectiveness of chemotherapy. An animal, 19 es?ecially a rat, capable of expressing a human isotype of 5-~ reductase (especially in the prostate gland) would be 21 highly desirable. ACAT is a key enzyme in lipid metabolism;
22 an animal model for its regulation would be extremely 23 valuableO Animals that express 5-lipoxygenase could be of 2~ interest to many research programs, particularly to screen isotype selective inhibitors. An animal which expressed 26 human hormone or cytokine receptor proteins would be 27 valuable in identifying agonists and antagonists of receptor 28 action~ Similarly, an animal that expressed components of 29 the human signal transduction system ~i.e. G proteins and phospholipase Cs, etc.) could be used to study the 31 pathophysiologic consequences of disordered function of 32 these proteins.
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J ~ r 1' ~c l The present invention can be used to produce cells and 2 animals which express human isotypes of transport proteins 3 (i.e. proteins which facilitate or enable the transport of 4 other molecules or ions across membranes in the gut, blood brain barrier, kidney, etc.). Such cells or animals can 6 then be used to study the role of such proteins in 7 metabolism. In particular, the extent and patterns of 8 conjugation mediated by such isotypes may be studied in 9 order to investigate the pharmacokinetic consequences of specifi~ differences in protein structure or sequence.
ll Glucoronide transferase, glycine conjugation and sulfation, 12 methylases, and glu~athione conjugation are examples of 13 enzymes of particular interest in this regard.
14 The clearance of many compounds is mediated by esterases. Cells or animals which express heterologous 16 isotypes of such esterases may be exploited in investigating 17 such clearance.
18 Cells or animals which express isotypes of proteins l9 involved in azo or nitro reduction would be desirable for research on the processes of azo or~nitro reduction.
21 Significantly, potential therapeutic agents are 22 ~requently found to induce toxic~effects in one animal model 23 but not in another animal model. To resolve the potential 24 of such agents, it is often necessary to determine the 2S metabolic patterns in various species, and to then determine 26 the toxicities of the metabolites. The present invention 27 permits one to produce transgenic cells or ~nimals which 28 could facilitate suoh determinations.
29 The methods of the present invention ~ay be used to produ~e alterations in a regulatory region ~or a native gene 31 sequence. Thus, the invention provides a means for altering 32 the nature or oontrol of transcription or translation of any 33 native gene sequence which is regulated by the regulatory .' : , ;. ,; , ' ' , .: '............................. ~
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l region. For example, it is possible to introduce mutations 2 which remove feedback inhibition, and thus result in 3 increased gene expression. Similarly, it is possible to 4 impair the transcriptional capacity of a sequence in order to decrease gene expression. Such alterations are 6 especially valuable in gene therapy protocols, and in the 7 development of improved animal models of human disease. For 8 example, the capacity to increas~ insulin gene transcription 9 or translation provides a potential genetic therapy for diabetes. Similarly, the ability to impair the synthesis of ll beta globln chains provides an animal model for beta-12 thalassemia.
13 The methods of the present invention, quite apart from 14 their uses in veterinary and human medicine, may be used to investiyate gene regulation, expression and organization in 16 animals.
l7 Since the methods of the present invention utilize 18 processes of DNA repair and recombination, agents which l9 inhibit or impair th~ present methods may act by affecting these processes. Since agents which impair DNA repair and 21 recombination have potential antineoplastic utility, the 22 present invention provides a 1Deans for identifying ~ovel 23 antineoplastic agents.
24 The present invention ~ay additionally be used to ~acilitate both the cloning of gene sequences, and the 26 mapping of chro~osomes or chromosomal abnormalities.
27 Since the desired gene sequence need not be homologous 28 or analogous to any native gene sequence of the recipient 29 c~ll, the methods of the present invention permit one to produce animals which contain and express foreign gene 31 sequences. I~ the cell expresses an analogous gene, the 32 desired gene sequence may be expressed in addition to such 33 analogous cellular genes (for ex~mple, an animal may express . , , . . . ~ . . . .

wv 91/1~7~ PCT/~S91/04006 t 1 both a "humani~ed" receptor and an analogous native 2 receptor). Thus, for example, t~e invention provides a 3 means for producing animals which express important human 4 proteins (Ruch as human interf~rons, tissue plasminogen acti~ator, hormones (such as insulin and growth hormone), 6 blood factors (such as Factor VIII), etc.).
7 In a second embodiment, the methods o~ the invention may 8 be used to introduce DNA into plant cells which can then be 9 manipulated in order to produc~ chimeric or transgenic ~0 plants. The plants which may be produced through 11 application of the disclosed method include all 12 multicellular, higher (i.e. non-fungal) plants. A non-13 fungal plant i5 any plant which is not a fungus or yeast.
14 In a third embodiment, the methods of the invention may be used to introduce DNA into the somatic cells of an animal 16 (particularly mammals including humans) or plant in order to 17 provide a treatment for genetic dise~se (i.e. "gene 18 therapy"). The principles of gene therapy are disclosed by 19 Oldham, R.X. (In: PrinciPles cf Biotherapy, Raven Press, NY, 1987), and similar texts.
21 In this third embodiment~ the genetic lesion which 22 causes the disease is replaced with a gene sequence encoding 23 a preferred gene product. Examples of such genetic lesions 24 axe those responsible for diseases such as cystic fibrosis, phenylketonuria, hemophilia, von Willebrand's Disease, 26 sickle cell anemia, thalasse~ia, galactosemia, fructose 27 intolerance, diseases of glycogen storage, hyper-28 cholesterolemia~ juvenile diabetes, hypothyroidism, 29 Alzheimer's Disease, Huntington's Disease, Gout, Lesch-Nyhan Syndrome, etc. (Bondy, P.R. et al., In: Disorde~s _of 31 Carbohydrate Metabolism, pp 221-340, Saunders Sl974);
32 Coleman, J. et al., Molecular Mechanisms of Disease, Yale 33 University Press, (1975)~. Disclosures of the methods and . ~ ~
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l uses for gene therapy are provided by Boggs, S.S. (Int. J.
2 Cell Clon. 8:80-96 (1990)); Karson, E.M. (Biol. Re~rod.
3 42:39-49 (l990)); Ledley, F.D., In: BiotechnoloqY A
4 Com~rehensive Treatise volume 7B, Gene Technolo~y, VCH
Publishers, Inc. NY, pp 399-458 (1989)); all of which 6 references are incorporated herein by reference.
7 In a fourth embodiment, the me~hods of the invention may B be used to provide a treatment to protect recipient animals 9 or plants from exposure to viruses, insects or herbicides (in the case of plants), insecticides, toxins, etc. In this 11 embodiment, the introduced gene would provide the recipient 1~ with gene sequences capable of mediating either an enhanced 13 or novel expression of an enzyme, or other protein, capable 14 of, for example, degrading an herbicide or toxin. For example, a plant cell may recaive a gene sequence capable of 16 mediating an enhanced or novel expression of a chitinase, 17 thus conferring increased resistance to insect parasites.
18 When providing the desired gene sequence to the cells of 19 an animal, pharmaceutically acceptable carriers (i.e.
liposomes, etc.) are preferably employed. Such gene 21 sequences can be formulated according to known methods to 22 prepare pharmaceutically useful compositions, whereby these 23 materials, or their functional derivatives, are combined in 24 admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, are 26 described, for example, in Nicolau, C. et al. (Crit. Rev.
27 Ther. Drua Carrier_Sy~t. 6:239-271 (1989)), which reference 28 is incorpor~ted herein by referenoe.
29 In order to form a pharmaceutically acceptable composition suitable for effective administration, such 31 compositions will contain an effective amount of the desired 32 gene sequence together with a suitable amount of carrier 33 vehicle.

: , ~ - ~ :: ~: . : , :
, WOs1/19796 PCT/~'S91/04006 ~; ^t~J,~ -58-1 Additional pharmaceutical methods may be employed to 2 control the duration of action. Control release 3 preparations may be ~chieved through the use of polymers to 4 complex or absorb the desired gene sequence (either with or S without any associated carrier). The controll~d deli~ery 6 may be exercised by selecting appropriate macromolecules 7 (for example polyesters, polyamino acids, polyvinyl, 8 pyrrolidone, ethylenevinylacetate, methylcellulose, 9 carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of ll incorporation in order to control relPase. Another possible 12 method to control the duration of action by controlled 13 release preparations is to incorporate the agent into 14 particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene 16 vinylacetate copolymers. Alternatively, instead of 17 incorporating these agents into polymeric particles, it is l8 possible to entrap these materials in microcapsules 19 prepared, ~or example, by coacervation techn~ques or by interfacial polymerization, for~example, hydroxymethylcellu-21 lose or gelatine-microcapsules and poly(methylmethacylate) 22 microcapsules, respectively, or in colloidal drug delivery 23 systems, for example, liposomes, albumin ~icrospheres, 24 microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
26 In a fifth embodiment, the methods of the present 27 invention may be used to improve the food or fiber 28 chara~teristics of plants or non-human animals. For 29 example, the methods can be used to increase the overall levels of protein synthesis thereby resulting in faster 3l growing plants or non-humah animals, or in the production of 32 plants and non-human animals which have increased food 33 value.

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-5~-1 Having now generally described the invention, the same 2 will be more readily understood through reference to the 3 following examples which are provided by way of 4 illustration, and are not intended to be limiting of the presPnt invention, unless specified.

7 E~AMPLE 1 8 ELECT~OPORATION : .

Electroporation was performed as follows: ~
11 . - .
12 DNA Preparation:

14 DNA used for electroporation was purified by CsCl gradient centrifugation. A large-scale digest of this 16 purified DNA was prepared by incubating the DNA with an 17 appropriate restriction enzyme. The large-scale digest was 18 examined for complete digestion by running 500 ~g on a 19 minigel. The DNA concentration of the large-scale digest ~
should be no higher than 1 ~g/~l. ` `
21 The large-scale digest was then extracted once with an 22 equal volume of phenol/chloroform and once with an equal 23 volume o~ chloroform. The DNA was precipitated with 2.4 24 volumes of ethanol, pelleted by centrifugation, and dried using a Speed-Vac. ;
26 The pelleted DNA was then resuspended at the desired 27 concentration (usually 1 ~g/~l) in a sterile Tris-EDTA
28 bu~fer such as O.lX TE (25 ~1 of DNA per electroporation~
29 The concentration of th~ DNA was then measured with a fluoro~eter.

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1 Preparation of Cells for_Electroporation:
3~mbryonic stem cells of the AB1 cell line were c~ltured 4to approximately 80% confluence according to the methods of SE.J. Robertson ~In: Teratocarcinomas and Embryonic Stem 6Cells: A Practical A~pr~ach, ~E.J. Robertson, Ed.), IRL
7Press, Oxford, 1987, pp 71-112). Cells were cultured in the 8presence of stomal cells which expressed lif into the 9culture medium. Cells were passaged 1:2 the day before 10electroporation, and fed 4 hours before harvesting.
11Cells were harvested by trypsinizing the cells, and by 12resuspending in media (cells from 2 x 10 cm plates were 13combined in a total volume of 10 ml in a 15 ml tube).
14The cells were pelleted by centrifugation, and the 15supernatant was removed by aspiration. The cells were then 16resuspended in 10 ml of phosphate buffered saline and the 17total number of cells was determined by counting a 20 ~
18aliquot. The usual yield is 30 x 106 cells per 10 cm plate.
19The cells were then pelleted by centrifugation and the 20supernatant was removed by aspiration. Cells were 21 resuspended at a density of 11 x 106 cells/ml. A 20 22 aliquot was counted to confir~ this cell density.

24 Electro~oration 26Cells, prepared as descri~ed above, were incubated in 27the presence of an appropriate amount of DNA in a 15 ml 2tube. 25 ~1 of DNA and 0.9 ml of cells were used for each 29el~ctroporation.
30The mixture was allowed to incubate at room temperature 31for 5 minutes (this step may, however, be omitted).
32The cell/DN~ mixture was then carefully aliquoted into 33electroporation cuvettes (O.~ ml per cuvette; the volume is , :`:

';' U'091/~9796 PCT/~'S91/04006 1 important). The cuvette was placed in the electroporation 2 holder with the foil electrodes in contact wi~h the metal 3 holding clips.
4 Electroporation was accomplished using a Biorad GenePulser set at 230V, 500 ~F (this requires a capacitance 6 extender). The time constant should read between 5.6 and ~ 7Ø
8 The cuvette was left at room temperature for 5 minutes 9 and then the cells were plated at an appropriate density (up to 2 x 1O7 cells/100 mm plate or 6 x lo6 cells/60 mm plate).
11 ~hen G418 was used as a selective agent, this cell density 12 should not be exceeded since G418 takes 3-4 days before 13 killing starts and plates will become over-confluent. When 14 G418 selection was to be applied, it is applied 24 hours post-electroporation. G418 concentration must be titrated 16 for every batch.
17 The plate(s) were re-fed with fresh media i G418 every 18 day for the first 6-7 days (until colonies are visible and 19 most cell debris has been removed). If usiny FIAU (0.2 ~M) ~election, this may proceed simultaneously.
21 The typical yield for RV4.0 (Thomas, K.R. et al., Cell 22 51:503-512 (1987)) is up to 104 colonies/107 cells/100 mm 23 plate. Although this yield may be significantly ~and 24 unpredictably) different from the yield obtained when other constructs are used, the use of the meth~d always results in 26 the recovery of some colonies of cells which contain the 27 electroporated DNA.
28 Colonies may be picked as early as 8 days. It is most 29 preferred to pick colonies at around 10-11 days. Colonies may, however, be recovered up to 18-21 days after t~e 31 electroporation.

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,, w~ `J~ 7Y~) PCI/I'S~1/0~006 1 EXAMPL~ 2 4 To illustrate the invention, embryonic stem ("~S") cells were co-electroporated with a 4.5 kb nptII-containing vector 6 (pPGXneobpA) which had been linearized by treatment with 7 XhoI restriction endonuclease, and with the 6.5 kb HPRT
8 vector, AD 8 (linearized with SacI) (Figure 7).
9 Electroporation (230 V, 500 ~F) were done on 0.9 ml aliquot of CCEp24 cells (7.5 x 106 cells/ml).
11 The electroporation reactions were conducted using molar 12 ratios of 1~ 10, and 1:100 (nptII DNA:HPRT DNA). The 13 total amount of DNA provided was either 25, 50, 100, or 200 14 ~9. The vectors used in this experiment are illustrated in ~', Figure 7. The results of this experi~ent are shown in Table ~-16 l. :-17 . :~
18 ~ TABLE 1 19 CO-ELECTROPORATION OF nptII ~ND hprt GENE SEQUENCES
21 Average of Number of Colonies Formed per 1 x 1o6 Cells ~
22 (~g of DNA (# = Number of trials averaged)) .. :
24 Ratio 200 100 50 25 of :-26 DNAG418R TGR #G418R TGR # G418R TGQ # G418R TGR # :
28 1:1233 2.7 3101 1.5 3 64 0 3 23 0 5 29 1:1046 0 316 0 5 8.7 0 7 nd nd :~
1:100 8 0.2 5 4.3 0 7 1.6 0 7 nd nd 32 This experiment shows that co-electroporation of an h~rt 33 gene sequence with an nptII-containing gene sequence in the 34 presence of selection ~or only the n~tII-containing sequence, resultad in recombination of both the nPtII and ~ -36 h~rt DNA molecules. ~

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1 The frequencies of recombination are shown in Table 2 2 below.

FREQUENCY OF RECOMBINATION `~

7 ExptRatio [DNA]G418R/10s TGR/107 TGR/G418R
8 Neo:Hprt ~g/ml A 1:1 20023.3 2.6 1/873 11 B 1:1 10010.1 1.0 1/1010 12 C 1:100 200 0.8 0.2 1/400* :
13 Cont --- 25 10.8 2.7 1/402 14 ~;~
15The reactions were carried out as described above. The 16reproducibility of the experimental results was examined.
17The results of this experiment are shown in Table 3.
1~
19 TABLE 3 ~:
20EFFECT OF MODIFIED CO-ELECTROPORATION PROTOCOL OM ~ :

23 Molar DNA # G418RIHPRT- HPRT- HPRT- G418R
24 ratios per of colonies G418R (per cell zap zap (total) transfected) ~ Neo:(~g) (X 10-9)(X 106) ~ .
28 1:1 200 816,150 / 32 1/504 400 202 :
29 100 3 3,030 / 3 1/1,010 100 105 S0 3 1,920 / 0 67 ~
31 25 5 1,150 / 0 24 ~:32 1:10 200 16 608 / 7 1/868 ` 43 47 :. : `
33 100 5 800 / 0 17 ::

1:100200 5 400 / 1 1/400 8 36 lO0 7 300 / 0 4.5 37 50 7 ~12 / 0 1.7 38 :::
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E~lPLE 3 2 HOMOLOGOI~S RECOMBIN~TION

4 In order to investigate the chromosomal structure ;
which is produced by the recombination of the vectors of the 6 above-described vectors into the chromosomes of recipient 7 cells, the following experiments were conducted.
8 For this purpose, a vector was used which contained a 9 6.5 kb region of homology with the cellular h~rt locus. The vector also contained the n~tII gene, as a selectable ma~er.
11 The vector was linearized with XhoI and provided to ES cells 12 by electroporation, as described above. Cells which became 13 resistant to G418 were selected and their DNA was analyzed --14 to determine if it contained restriction fragments that were lS consistent with the predicted integration structure.
16 The vector used, and the predicted integration structure 17 are illustrated in Figure 8. Gel electrophoresis of 18 restriction digests of cellular DNA confirmed that the G418 ~9 resistant cells contained the bE~rt structure shown in Figure );
8. This finding confirmed that the vector had integrated 21 into the chromosome of the cell by homologous recombination 22 at the hPrt locus. -~

~XAMPL~ 4 26 REVERSION OF RECOMaINAN~8 ~-~
27 ~ ~
28 The effect of the size of the region of homology carried ~ -.
29 by the vector-on the reversion frequency of recombinants was deter~ined. Recombinants containing a vector having 6.8 kb 31 of homology with the h~rt locus were prepared as described 32 - in Example 3. Using the same method, reco~binants were also 33 prepared which contained a similar vector having only 1.3 kb ;~
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WO 91/19796 PCr/l:'S91/04006 1 of homology with the hprt locus. The structures of the 2 insertion site of the 6.~ kb vector ls illustrated in Figure 3 8. The reversion frequency of the two constructs is shown 4 in Table 4. The structure obtained from the reversion of the insertion is shown in Figure 9.

Du~lication~ Clones ~ Revertible Freauencv xlO-s 12 6.8 kb l9 19 3.3 to 0.2 13 1.3 kb 2 2 1.2 to 0.3 16 E~MPLE 5 17 TARGETING FREO~ENCY OF :~NSERTION AND PcEPL~CEMENT VECTORS ~:

19 A series of different Yectors were used to investigate the targeting freguency achieved through the use of the ~;
21 methods of the invention. These vectors contained 6.8 kb of 22 homology with the murine h~rt gene and had regions of 23 heterology either at the linearization site or internally 24 (Figure 2).
For this pur~ose, 108 cells were electroporated into ES
26 cells, prepared as described above, and plated onto 10 x 90 27 mm plates. After 24 hours G418 (at 350 ~g/ml) was added to 28 the media. After 5 days ~election 105 M 6-thioguanine was 29 added to 9 plates, 1 was retained under G418 selection as the transfection control. Selection was continued for an 31 additional 7 days. Colo~ies were scored at this time and 32 expanded ~or south~rn analysis as separate clones.
33 Targetiny efficiencies are detailed for each of the vectors 34 IFigur~ 2; Table 5).

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1 Southern analysis showed that the majority of the 6-TGR
2 clones had the predicted integration structure depicted for 3 HindIII digestion in Figure 8.
4 Reversion of the hprt clones was done by measuring HATR.
Cells were clonally expanded under 6-TG selection o prevent 6 "jackpot" effects cau~ed by the early recombinational loss 7 of the duplicated element giving rise to a large number of 8 colonies by cell division. When 107 cells were obtained, the 9 cells were reseeded onto 90 mm plates without selection for 48 hours. After 48 hours HAT selection was applied and 11 resistant colonies were scored 10 days later, typically 20 12 to 200 colonies were observed per 107 cells plated (Table 4).
13 Every clone examined reverted at a similar frequency.

16 REPLACEMENT AND INSERTION VECTORS: TARGETING AND FREQUENCY

18 Gene Homoloov Vector Frequencv 19 -:
~prt 6.8 kb RV 1/300 ~ 10X
21 ~prt 6.8 kb IV 1/32 23 Hprt 1.3 kb RV <1/5000 24 minimum 1 12x :.
Hprt 6.8 kb IV 1/400 -~7 Hox2.6 3.2 kb IV+ 1/33 28 :
29 RV=Replacement Vector; IV=Insertion Vector ~ ~
. ~ :

34 EXA~P~E 6 6ELECTION FOR HO~O~OGO~8 ~ECOM~INATION
36 .
37 It is possible to use "Poly A Selection" in order to ~ -38 enhance the selection of cells which have integrated the 39 introduced DNA by homologous recombination.

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1 If an introduced DNA molecule were to integrate at 2 random into the host chromosoI~e, it would generally ~ot 3 integrate at a site adjacent to a necessary 3' 4 polyadenylation site. Thus, the mRNA produced by the transcription of sùch randomly inserted constructs would 6 generally lack polyadenylation. This fact can be exploited 7 by using vectors which permit one to select for a 8 recombinational event that results in integration adjacent 9 to the natural polyadenylation site of the introduced gene sequence (i.e. by homologous recombination rather than by 11 random insertion).
12 To illustrate this aspect of the invention, three 13 vectors were constructed which contain fragments of the hprt 14 gene (Figure lO). As shown in Fiqure 10, the vectors contain exons 7, 8, and 9 of the hPrt gene. The 16 polyadenylation site is located in exon 9. A ~inDIII site 17 is present within exon 9, and an EcoRI site is located after 18 the end of the exon.
19 The first vectoE em~)loyed contained a 5.0 kb region, and thus contained the polyadenylation site of exon 9 (Vector 6, 21 Figure 10). As shown in Table 6, the frequency of insertion 22 was high (i.e. frequency of G418 resistant colonies was 24 23 x 105), but only 1/941 colonies showed the dual thioguanine 24 resistance and G418 resistance which would characterize a desired recombinant (i.e. a recombinant in which integration 26 had resulted in an intact h~rt gene and an i~tact n~tII
27 gene)0 Thus, some random integration is occurring.
28 Similarly, when a vector of 3.5 kb was employed (Vector 29 lO) which contained DNA from the XbaI site to the coRI site of Vector 6, the ~reguency of insertion was high (i.e.
31 frequency of G418 resistant colonies was 21 x 105), but only 32 1/770 colonies showed the dual thioguanine resistance and 33 G418 resistance which would characterize a desired . .

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.~ - ~ . , . -- ~ .': . ~, :, W091~19796 PCT/~!S91/04006 l recombinant (Table 6). This findlng demonstrates that some 2 random integration is occurring.
3 ~f, however, a vector is employed which lacks the 4 polyadenylation site of exon 9 (i.e. Vector 9), random integration does not result in expression of a functional 6 nptII transcript. Thus, the frequency of G418 resistant 7 colonies is low ( 1 . 4 X lo-5? . Since the number of colonies 8 evidencing random integratio~ is suppressed, the overall 9 frequency of recovery of the desired recombinants is enhanced (i.e. an overall ef~iciency of l/lOO for the dual ll resistant colonies (Table 6). Thus, the poly A selection 12 results in an approximate increase of overall efficiency of 13 nearly lO fold. Poly A selection may therefore ~e ;;
14 advantageously used in situations where one desires to lS minimize or avoid the screening of colonies to identify ~' 16 random versus homologous recombinants.
17 , ;
l8 TABLE 6 ~--l9 POLY A SELFCTION -2.1 VECTOR SIZE G418R TGR TGR/G418R
22 # (kb) (x lO-s) (x 107) 24 6 5.0 24 2.S l/941 ~ ~ -9 3.0 l.4 l.4 l/lOO
26 lO 3.5 21 2.7 l/770 27 -~-E2A~P~E 7 31 ~NTROD~TION OF 8~T~E ~TATION8 IN ~RE C-8RC ~0~8 33 ~he methods of the present invention were further 34 illustrated by their use to produce cells having precise and subtle mutations in the c-src locus of ES cells. The c-src ~-36 locus contains several exons, which are designated as 37 ~'boxed" regions 2 and 3' in Figure ll. As shown in Figure ~.

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WO 91/19~96 PCrt~lS91/04006 7~3~
-6~-1 llA, the natural allele of exon 3' does not contain a 2 HindIII site.
3 The sequence of a portion of exon 3' is shown in Figure 4 llC. As shown in Figure llC, a 9 bp i~sertion into ~his exon will result in the formation of a HinDI~I site.
6 To accomplish this change in the sequence of exon 3', a 7 vector (src 14) was prepared. As shown in Figure llB, the 8 src 14 vector is homologous to a region of the c-src locus.
9 The exon 3' sequence of the vector, howe~er, has been altered to contain the 9 base pair insertion needed to 11 creat~ a HindIII site (Figure llC).
12 The src 14 vector was introduced into E5 cells by co-13 electroporation with a second vector (PGKneo) that oontained 14 the ~E~I gene, at a total DNA concentration of 25 ~g/ml and a molar ratio of 1:5 (neo vector to targeting vector) in the 16 manner described above.
17 Cells were cultured in the presence of G~18 for 12 days 18 in order to select for recombinant cells in which the nptII
19 gene had integrated. These recom~inant cells were then screened, using PCR, for cells which had undergone a 21 recombinational event resulting in the replacement of the 22 natural exon 3' locus with the HinDIII site-containing exon 23 3' sequence of the src 14 vector.
24 Southern analysis of the colonies identified by PCR
screening using probes B and C (Figure llB) demonstrated 26 that the natural exon 3' locus had been altered, as de~ired, 27 to contain a HinDIII site. This experiment demonstrated 28 that subtle insertions can be introduced into any`cellular 29 gene.
To further illustrate the capacity o~ the present 31 invention to introduce complex, predetermined mutations into 32 the geno=e of a recipient cell, exon 3" ol the c-sr~ gene of ~ ' WO9l/19796 PCT/~'S91/04006 1 an ES cell was mutated to contain two different substitution 2 mutations.
3 As shown in Figure 12A, the natural allele of exon 3"
4 does not contain either an NheI site or an EcoRI site. As shown in Figure 12C, howe~er, thP replacement of the natural 6 sequence ACC TGG TTC of exon 3" with the sequence TAG CTA
7 GCT will result in the formation of an NheI site.
8 Similaxly, replacement of ACA with GAA in exon 3" will 9 create an EcoRI site (Figure 12C).
To accomplish these changes in the sequence of exon ", 11 a vector (src 33) was prepared. As shown in Figure 12B, the 12 src 33 vector is homologous to a region of the c-src locus.
13 The exon 3" sequence of the vector, however, has been 14 altered to c~ntain the substitutions indicated above (Figure 12C).
16 The src 33 vector was introduced into E5 cells by 17 electroporation, in concert with a second vector that 18 contained the nptII gene, in the manner described above.
19 Cells were cultured in the presence of G418 in order to select for recombinant cells in which the n~tII gene had 21 integrated. These recombinant cells were then screened, 22 using PCR, for cells which had undergone a second 23 recombinational event resulting in the replacement of the 24 natural exon 3" locus with the exon 3" sequence of the src 33 vector.
26 Southern analysis of the colonies identified by PCR
27 screening using probes A and C ~Figure 12C) demonstrated 28 that the natural exon 3" locus had been altered, as desired, 29 to contain both the NheI and the EcoRI sites. This experiment demonstrated that subtle substitutions can be ~
31 introduced into any cellular gene. -32 While the invention has been described in connection 33 with specific embodiments thereof, it will be understood .
~: :: `' , W091/19796 PCT/~'S9l/04006 1 that it is capable of further modifications and this 2 application is intended to cover any variations, uses, or 3 adaptations of the invention following, in general, the 4 principles of the invention and including such departures from the present disclosure as come within known or 6 customary practice within the art to which the invention 7 pertains and as may be applied to the essential features 8 hereinbefore set forth and as follows in the scope of the 9 appended claims.

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Claims (37)

WHAT IS CLAIMED IS:

1. A method for obtaining a desired animal or non-fungal plant cell which contains a desired non-selectable gene sequence inserted within a predetermined gene sequence of said cell's genome, which method comprises:
A. incubating a precursor cell with a DNA molecule containing said desired non-selectable gene sequence, wherein said DNA molecule additionally contains two regions of homology which flank said desired gene sequence, and which are sufficient to permit said desired gene sequence to undergo homologous recombination with said predetermined gene sequence of said genome of said precursor cell;
B. causing said DNA molecule to be introduced into said precursor cell;
C. permitting said introduced DNA molecule to undergo homologous recombination with said predetermined gene sequence of said genome of said precursor cell to thereby produce said desired cell wherein said desired non-selectable gene sequence has been inserted into said predetermined gene sequence; and D. recovering said desired cell.

2. The method of claim 1 wherein said DNA molecule contains a detectable marker gene sequence.

3. The method of claim 1 wherein said DNA molecule is introduced into said precursor cell by subjecting said precursor cell and said DNA molecule to electroporation.

4. The method of claim 3 wherein in step B, said precursor cell is simultaneously subjected to electroporation with a second DNA molecule, said second DNA
molecule containing a detectable marker gene sequence.

5. The method of claim 1 wherein said desired cell is a non-fungal plant cell.

6. The method of claim 1 wherein said desired cell is an animal cell.

7. The method of claim 6 wherein said animal cell is a somatic cell.

8. The method of claim 7 wherein said animal sell is of an animal selected from the group consisting of a chicken, a mouse, a rat, a hamster, a rabbit, a sheep, a goat, a fish, a pig, a cow or bull, a non-human primate and a human.

9. The method of claim 6 wherein said animal cell is a pluripotent cell.

10. The method of claim 9 wherein said animal cell is of an animal selected from the group consisting of a chicken, a mouse, a rat, a hamster, a rabbit, a sheep, a goat, a fish, a pig, a cow or bull, and a non-human primate.

11. The method of claim 9 wherein said pluripotent cell is an embryonic stem cell.

12. The method of any one of claims 1-3 wherein said desired gene sequence is substantially homologous to said predetermined gene sequence of said precursor cell.

13. The method of claim 12 wherein said desired gene sequence is an analog of said predetermined sequence of said precursor cell.

14. The method of claim 12 wherein said desired gene sequence is a human analog of said predetermined sequence of said precursor cell.

15. The method of claim 12 wherein said desired cell is a non-human cell which expresses said desired gene sequence.

16. The method of claim 12 wherein said desired gene sequence encodes a protein selected from the group consisting of: a hormone, an immunoglobulin, a receptor molecule, a ligand of a receptor molecule, and an enzyme.

17. A non-fungal plant cell which contains an introduced recombinant DNA molecule containing a desired gene sequence, said desired gene sequence being flanked by regions of homology which are sufficient to permit said desired gene sequence to undergo homologous recombination with a predetermined gene sequence of the genome of said cell.

18. A non-human animal cell which contains an introduced recombinant DNA molecule containing a desired gene sequence, said desired gene sequence being flanked by regions of homology which are sufficient to permit said desired gene sequence to undergo homologous recombination with a predetermined gene sequence of the genome of said c?ll.

19. The desired cell produced by the methods of any one of claims 1-3.

20. The desired cell produced by the method of claim 11.

21. The desired cell produced by the method of claim 12.

22. A non-human animal containing a cell derived from the desired cell of claim 19, wherein said animal is either a chimeric or a transgenic animal.

23. The non-human animal of claim 22, wherein said animal and said desired cell are of the same species, and wherein said species is selected from the group consisting of: a chicken, a mouse, a rat, a hamster, a rabbit, a sheep, a goat, a fish, a pig, a cow or bull, and a non-human primate.

24. A non-human animal containing a cell derived from the desired cell of claim 20, wherein said animal is either a chimeric or a transgenic animal.

25. The non-human animal of claim 24, wherein said animal and said desired cell are of the same species, and wherein said species is selected from the group consisting of: a chicken, a mouse, a rat, a hamster, a rabbit, a sheep, a goat, a fish, a pig, a cow or bull, and a non-human primate.

26. A non-human animal containing a cell derived from the desired cell of claim 21, or a descendant thereof, wherein said animal is either a chimeric or a transgenic animal.

27. The non-human animal of claim 26, wherein said animal and said desired cell are of the same species, and wherein said species is selected from the group consisting of: a chicken, a mouse, a rat, a hamster, a rabbit, a sheep, a goat, a fish, a pig, a cow or bull, and a non-human primate.

28. A non-fungal plant containing a cell derived from the desired cell of claim 5, or a descendant thereof, wherein said non-fungal plant is either a chimeric or a transgenic plant.

29. A method of gene therapy which comprises introducing to a recipient in need of such therapy, a desired non-selectable gene sequence, said method comprising:
A. providing to said recipient an effective amount of a DNA molecule containing said desired non-selectable gene sequence, wherein said DNA molecule additionally contains two regions of homology which flank said desired gene sequence, and which are sufficient to permit said desired gene sequence to undergo homologous recombination with a predetermined gene sequence present in a precursor cell of said recipient;
B. permitting said DNA molecule to be introduced into said precursor cell;
C. permitting said introduced DNA molecule to undergo homologous recombination with said predetermined gene sequence of said genome of said precursor cell to thereby produce a desired cell wherein said desired non-selectable gene sequence has been inserted into said predetermined gene sequence; and wherein the presence or expression of said introduced gene sequence in said cell of said recipient comprises said gene therapy.

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

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

32. The method of claim 31 wherein said animal is selected from the group consisting of: a chicken, a mouse, a rat, a hamster, a rabbit, a sheep, a goat, a fish, a pig, a cow or bull, a non-human primate and a human.

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

34. A method for obtaining a desired animal or non-fungal plant cell which contains a desired non-selectable gene -sequence inserted within a predetermined gene seguence of said cell's genome, which method comprises:
A. incubating a precursor cell under non-selective culture conditions, or under a first set of selective culture conditions, with a DNA molecule containing:
i) said desired non-selectable genP sequence, wherein said DNA mo:Lecule additionally contains two regions of homology which flank said desired gene sequence, and which are sufficient to permit said desired gene sequence to undergo homologous recombination with said predetermined gene sequence of said genome of said precursor cell;
and ii) a selectable gene sequence whose presence or expression in said precursor cell can be selected for by culturing said cell under said first set of selective culture conditions, and whose presence or expression in said precursor cell can be selected against by culturing said cell under a second set of selective culture conditions;
B. permitting said DNA molecule to be introduced into said precursor cell;
C. permitting said introduced DNA molecule to undergo homologous recombination with said predetermined gene sequence of said genome of said precursor cell to thereby produce said desired cell wherein said desired non-selectable gene sequence has been inserted into said predetermined gene sequence; and D. recovering said desired cell by culturing said cell under said first set of selective culture conditions, by then permitting said cell to undergo intrachromosomal recombination under non-selective culture conditions, and by then incubating said cell under said second set of selective culture conditions.

35. The method of claim 34, wherein said cell is deficient in HPRT enzyme, and wherein said selectable gene sequence expresses an active HPRT enzyme, and wherein said first set of selective culture conditions comprises incubation of said cell under conditions in which the presence of an active HPRT enzyme in said cell is required for growth, and wherein said second set of selective culture conditions comprises incubation of said cell under conditions in which the absence of an active HPRT enzyme in said cell is required for growth.

36. The method of claim 34, wherein said cell is deficient in APRT enzyme, and wherein said selectable gene sequence expresses an active APRT enzyme, and wherein said first set of selective culture conditions comprises incubation of said cell under conditions in which the presence of an active APRT enzyme in said cell is required for growth, and wherein said second set of selective culture conditions comprises incubation of said cell under conditions in which the absence of an active APRT enzyme in said cell is required for growth.

37. The method of claim 34, wherein said cell is deficient in TK enzyme, and wherein said selectable gene sequence expresses an active TK enzyme, and wherein said first set of selective culture conditions comprises incubation of said cell under conditions in which the presence of an active TK enzyme in said cell is required for growth, and wherein said second set of selective culture conditions comprises incubation of said cell under conditions in which the absence of an active TK enzyme in said cell is required for growth.