WO1993025674A1

WO1993025674A1 - Non-human animals having retinoblastoma gene alterations

Info

Publication number: WO1993025674A1
Application number: PCT/US1993/005584
Authority: WO
Inventors: Allan Bradley; Wen-Hwa Lee; Eva Y.-H. P. Lee
Original assignee: Baylor College Of Medicine; The Regents Of The University Of California
Priority date: 1992-06-11
Filing date: 1993-06-10
Publication date: 1993-12-23
Also published as: AU4632293A

Abstract

A desired non-human animal or an animal cell or human cell which contains a predefined alteration in at least one of its two chromosomal rb alleles, such that the expression of at least one of these alleles is altered.

Description

TIT E OF THE INVENTION:

NON-HUMAN ANIMALS HAVING -RETINOBLASTOMA GENE

ALTERATIONS

FIELD OF THE INVENTION;

The invention is directed toward non-human animals having predefined, alterations in a chromosomal allele of a retinoblastoma gene. The invention further pertains to the use of such animals in the development of agents and therapies for tumor cells having a retinoblastoma gene deficiency. The invention was made with Government support under Grant Nos. CA-49649 and EY-05758 awarded by the National Institutes of Health. The Government has certain rights in this invention.

CROSS-REFERENCE TO RELATED APPLICATIONS:

This application is a continuation-in-part of U.S. Patent Application Serial Nos. 07/637,563 (filed January 4, 1991), and 07/816,740 (filed January 3, 1992). This application claims priority to U.S. Patent Application, Serial No. 07/897,134, which is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION:

I. Chimeric and Transgenic Animals

Recent advances in recombinant DNA and genetic technologies have made it possible to introduce and express a desired gene sequence in a recipient animal.

Through the use of such methods, animais have been engineered to contain gene sequences that are not normally or naturally present in an unaltered animal. The techniques have also been used to produce animals which exhibit altered expression of naturally present gene sequences. The animals produced through the use of these methods are known as either "chimeric" or "transgenic" animals. Such animals contain cells that have been genetically altered, and do not arise spontaneously in nature. In a "chimeric" animal, only some of the animal's cells contain and express the introduced gene sequence, whereas other cells have been unaltered. The capacity of a chimeric animal to transmit the introduced gene sequence to its progeny depends upon whether the introduced gene sequences are present in the germ cells of the animal. Thus, only certain chimeric animals can pass along the desired gene sequence to their progeny.

In contrast, all of the cells of a "transgenic" animal contain the introduced gene sequence. Conse¬ quently, every transgenic animal is capable of transmitting the introduced gene sequence to its progeny.

II. Production of Transgenic Animals: Microinjection Methods

The most widely used method through which transgenic animals have been produced involves injecting a DNA molecule into the male pronucleus of a fertilized egg (Brinster, R.L. et al.. Cell 22:223 (1981); Costantini, F. et al.. Nature 294:92 (1981); Harbers, K. et al.. Nature 293:540 (1981); Wagner, E.F. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 7_8.:5016 (1981) ; Gordon, J.W. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 73:1260 (1976); Stewart, T.A. et al.. Science 217:1046-1048 (1982); Palmiter, R.D. et al. , Science 222:809 (1983); Evans, R.M et al. (U.S. Patent 4,870,009).

The gene sequence being introduced need not be incorporated into any kind of self-repiicating plasmid or virus (Jaenisch, R. , Science. 240:1468-1474 (1988)). Indeed, the presence of vector DNA has been found, in many cases, to be undesirable (Hammer, R.E. et al.. Science 235:53 (1987); Chada, K. et al.. Nature 319:685 (1986); Kollias, G. et al.. Cell 46:89 (1986); Shani, M. , Molec. Cell. Biol. 6:2624 (1986); Chada, K. et al.. Nature 314:377 (1985); Townes, T. et al.. EMBO J. 4:1715 (1985)).

After being injected into the recipient fertilized egg, the DNA molecules are believed to recombine with one another to form extended head-to-tail concatemers. It has been proposed that such concatemers occur at sites of double-stranded DNA breaks at random sites in the egg's chromosomes, and that the concatemers are inserted and integrated into such sites (Brinster, R.L. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 82:4438 (1985)). Although it is, thus, possible for the injected DNA molecules to be incorporated at several sites within the chromosomes of the fertilized egg, in most instances, only a single site of insertion is observed (Jaenisch, R. , Science. .240:1468-1474 (1988)).

Once the DNA molecule has been injected into the fertilized egg cell, the cell is implanted into the uterus of a recipient female, and allowed to develop into an animal. Since all of the animal's cells are derived from the implanted fertilized egg, all of the cells of the resulting animal (including the germ line cells) shall contain the introduced gene sequence. If, as occurs in about 30% of events, the first cellular division occurs before the introduced gene sequence has integrated into the cell's genome, the resulting animal will be a chimeric animal.

By breeding and inbreeding such animals, it has been possible to produce heterozygous and homozygous transgenic animals. Despite any unpredictability in the formation of such transgenic animals, the animals have generally been found to be stable, and to be capable of producing offspring which retain and express the introduced gene sequence.

Since microinjection causes the injected DNA to be incorporated into the genome of the fertilized egg through a process involving the disruption and alteration of the nucleotide sequence in the chromosome of the egg at the insertion site, it has been observed to result in the alteration, disruption, or loss of function of the endogenous egg gene in which the injected DNA is inserted. Moreover, substantial alterations (deletions, duplications, rearrangements, and translocations) of the endogenous egg sequences flanking the inserted DNA have been observed (Mahon, K.A. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 5:1165 (1988); Covarrubias, Y. et al.. Proc. Natl. Acad. Sci. (U.S.A.) -82:6020 (1986); Mark, W. et al.. Cold Spr. Harb. Symp. Quant. Biol. .50:453 (1985)). Indeed, lethal mutations or gross morphological abnormalities have been observed (Jaenisch, R. , Science 240:1468-1474 (1988); First, N.L. et al.. Amer. Meat Sci. Assn. 39th Reciprocal Meat Conf. 39:41 (1986)).

Significantly, it has been observed that even if the desired gene sequence of the microinjected DNA molecule is one that is naturally found in the recipient egg's genome, integration of the desired gene sequence rarely occurs at the site of the natural gene (Brinster, R.L. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 86:7087-7091 (1989)). Moreover, introduction of the desired gene sequence does not generally alter the sequence of the originally present egg gene. Although the site in the fertilized egg's genome into which the injected DNA ultimately integrates cannot be predetermined, it is possible to control the expression of the desired gene sequence such that, in the animal, expression of the sequence will occur in an organ or tissue specific manner (reviewed by Westphal, H. , FASEB J. 2:117 (1989); Jaenisch, R. , Science 240:1468- 1474 (1988); Meade, H. et al. (U.S. Patent 4,873,316)). The success rate for producing transgenic animals is greatest in mice. Approximately 25% of fertilized mouse eggs into which DNA has been injected, and which have been implanted in a female, will become transgenic mice. A lower rate has been thus far achieved with rabbits, sheep, cattle, and pigs (Jaenisch, R. , Science 240:1468- 1474 (1988); Hammer, R.E. et al.. J. Animal. Sci. 63:269 (1986); Hammer, R.E. et al.. Nature 315:680 (1985); Wagner, T.E. et al.. Therioqeno1oqy 2-1:29 (1984)).- The lower rate may reflect greater familiarity with the mouse as a genetic system, or may reflect the difficulty of visualizing the male pronucleus of the fertilized eggs of many farm animals (Wagner, T.E. et al.. Therioqenoloqy 21:29 (1984)). Thus, the production of transgenic animals by microinjection of DNA suffers from at least two major drawbacks. First, it can be accomplished only during the single-cell stage of an animal's life. Second, it requires the disruption of the natural sequence of the DNA, and thus is often mutagenic or teratogenic (Gridley, T. et al.. Trends Genet. 3:162 (1987)).

III. Production of Chimeric and Transgenic Animals: Recombinant Viral and Retroviral Methods

Chimeric and transgenic animals may also be produced using recombinant viral or retroviral techniques in which the gene sequence is introduced into an animal at a multi-s' .--11 stage. In such methods, the desired gene sequence is introduced into a virus or retrovirus. Cells which are infected with the virus acquire the introduced gene sequence. If the virus or retrovirus infects every cell of the animal, then the method results in the production of a transgenic animal. If, however, the virus infects only some of the animal's cells, then a chimeric animal is produced. The general advantage of viral or retroviral methods of producing transgenic animals over those methods which involve the microinjection of non-replicating DNA, is that it is not necessary to perform the genetic manipulations at a single cell stage. Moreover, infection is a highly efficient means for introducing the DNA into a desired cell.

Recombinant retroviral methods for producing chimeric or transgenic animals have the advantage that retroviruses integrate into a host's genome in a precise manner, resulting generally in the presence of only a single integrated retrovirus (although multiple insertions may occur) . Rearrangements of the host chromosome at the site of integration are, in general, limited to minor duplications (4-6 base pairs) of host DNA at the host virus junctions (Jaenisch, R. , Science 240:1468-1474 (1988); see also, Varmus, H., In: RNA Tumor Viruses (Weiss, R. et al.. Eds.), Cold Spring Harbor Press, Cold Spring Harbor, NY, pp. 369-512 (1982)). The method is, however, as mutagenic as microinjection methods.

Chimeric animals have, for example, been produced by incorporating a desired gene sequence into a virus (such as bovine papilloma virus or polyoma) which is capable of infecting the cells of a host animal. Upon infection, the virus can be maintained in an infected cell as an extrachromosomal episome (Elbrecht, A. et al.. Molec. Cell. Biol. : 276 (1987); Lacey, M. et al.. Nature 322:609 (1986); Leopold, P. et al.. Cell 51:885 (1987)). Although this method decreases the mutagenic nature of chimeric/transgenic animal formation, it does so by decreasing germ line stability, and increasing oncogenicity.

Pluripotent embryonic stem cells (referred to as "ES" cells) are cells which may be obtained from embryos until the early post-implantation stage of embryogenesis. The cells may be propagated in culture, and are able to differentiate either in vitro or in vivo upon implantation into a mouse as a tumor. ES cells have a normal karyotype (Evans, M.J. et al.. Nature 292:154-156 (1981) ; Martin, G.R. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 78:7634-7638 (1981)).

Upon injection into a blastocyst of a developing embryo, ES cells will proliferate and differentiate, thus resulting in the production of a chimeric animal. ES cells are capable of colonizing both the somatic and germ-line lineages of such a chimeric animal (Robertson, E. et al.. Cold Spring Harb. Conf. Cell Prolif. 10:647- 663 (1983); Bradley A. et al.. Nature 309:255-256 (1984); Bradley, A. et al. _r Curr. Top. Devel. Biol. 20:357-371 (1986); Wagner, E.F. et al.. Cold Spring Harb. Svmp. Quant. Biol. 50:691-700 (1985); (all of which references are incorporated herein by reference) .

In this method, ES cells are cultured in vitro, and infected with a viral or retroviral vector containing the gene sequence of interest. Chimeric animals generated with retroviral vectors have been found to have germ cells which either lack the introduced gene sequence, or contain the introduced sequence but lack the capacity to produce progeny cells capable of expressing the introduced sequence (Evans, M.J. et al.. Cold Spring Harb. Symp. Quant. Biol. 50:685-689 (1985); Stewart, CL. et al.. EMBO J. 4:3701-3709 (1985); Robertson, L. et al.. Nature (1986) ; which references are incorporated herein by reference) .

Because ES cells may be propagated in vitro, it is possible to manipulate such cells using the techniques of somatic cell genetics. Thus, it is possible to select ES cells which carry mutations (such as in the hprt gene (encoding hypoxanthine phosphoribosyl transferase) (Hooper, M. et al.. Nature 326:292-295 (1987) ; Kuehn, M.R. et al.. Nature 326:295-298 (1987)). Such selected cells can then be used to produce chimeric or transgenic mice which fail to express an active HPRT enzyme, and thus provide animal models for diseases (such as the Lesch-Nyhan syndrome which is characterized by an HPRT deficiency) .

As indicated above, it is possible to generate a transgenic animal from a chimeric animal (whose germ line cells contain the introduced gene sequence) by inbreeding.

The above-described methods permit one to screen for the desired genetic alteration prior to introducing the transfected ES cells into the blastocyst. One drawback of these methods, however, is the inability to control the site or nature of the integration of the vector.

IV. Production of Chimeric and Transgenic Animals: Plasmid Methods

The inherent drawbacks of the above-described methods for producing chimeric and transgenic animals have caused researchers to attempt to identify additional methods through which such animals could be produced.

Gossler, A. et al.. for example, have described the use of a plasmid vector which had been modified to contain the gene for neomycin phosphotransferase (nptll gene) to transfect ES cells in culture. The presence of the nptll gene conferred resistance to the antibiotic

G418 to ES cells that had been infected by the plasmid

(Gossler, A. et al.. Proc. Natl. Acad. Sci. (U.S.A.)

22:9065-9069 (1986), which reference is incorporated herein by reference) . The chimeric animals which received the plasmid and which became resistant to G418, were found to have integrated the vector into their chromosomes.

Takahashi, Y. et al. have described the use of a plasmid to produce chimeric mice cells which expressed an avian crystallin gene (Development 102:258-269 (1988), incorporated herein by reference) . The avian gene was incorporated into a plasmid which contained the nptll gene. Resulting chimeric animals were found to express the avian gene.

V. Introduction of Gene Sequences into Somatic Cells

DNA has been introduced into somatic cells to produce variant (chimeric) cell lines. hprt-deficient Chinese hamster ovary (CHO) cells have been transformed with the CHO hprt gene in order to produce a prototrophic cell line (Graf, L.H. et al.. Somat. Cell Genet. 5:1031- 1044 (1979)). Folger et al. examined the fate of a thymidine kinase gene (tk gene) which had been microinjected into the nuclei of cultured mammalian cells. Recipient cells were found to contain from 1 to 100 copies of the introduced gene sequence integrated as concatemers at one or a few sites in the cellular genome (Folger, K.R. et al.. Molec. Cell. Biol. 2:1372-1387 (1982)). DNA-mediated transformation of an RNA polymerase II gene into Syrian hamster cells has also been reported (Ingles, C. et al.. Molec. Cell. Biol. 2:666-673 (1982)).

Plasmids conferring host neomycin resistance and guanosine phosphotransferase activity have been transfected into Chinese hamster ovary cells to generate novel cell lines (Robson, C.N. et al.. Mutat. Res. 162:201-208 (1986)).

VI. Oncogenes and tumor suppressor genes

One mechanism through which cancer may arise is through a cell's exposure to a carcinogenic agent, either chemical or radiation. Such exposure may damage the DNA sequence of critical genes present in the genome of a cell of an animal. If this damage leads to either an impairment in the expression of the gene, or in the production of a mutant gene product, the cell may then proceed to proliferate, and ultimately result in the formation of a tumor.

One class of such critical genes^ has been referred to as "oncogenes." Oncogenes are genes which are naturally in an "inactivated" state, but which, through the effect of the DNA damage are converted to an "activated" state capable of inducing tumorigenesis (i.e., tumor formation). Oncogenes have been identified in 15-20% of human tumors. The products of oncogenes ("oncoproteins") can be divided into two broad classes according to their location in the cell.

Oncogene products which act in the cytoplasm of cells have readily identifiable biochemical or biological activities (Green, M.R. , Cell 56:1-3 (1989)). Those that act in the nucleus of a cell have been more difficult to characterize. Some nuclear oncoproteins (such as E1A and mvc) have transcriptional regulatory activity, and are believed to mediate their activities by the transcriptional activation of cellular genes (Kingston, R.E., Cell 4.1:3-5 (1985)). Other nuclear oncoproteins appear to have a complex array of activities (such as DNA binding activity, ability to initiate viral DNA synthesis, ATPase activity, helicase activity, and transcriptional regulatory activity) (Green, M.R. , Cell 56:1-3 (1989)).

The creation of a mutant oncogene is only one of the requirements needed for tumor formation; tumorigenesis appears to also require the additional inactivation of a second class of critical genes: the "anti-oncogenes" or "tumor-suppressing genes." In their natural state these genes act to suppress cell proliferation. Damage to such genes leads to a loss of this suppression, and thereby results in tumorigenesis. Thus, the deregulation of cell growth may be mediated by either the activation of oncogenes or the inactivation of tumor-suppressing genes (Klein, G., Science 238:1539-1545 (1987); Weinberg, R.A. , Scientific A er.. Sept. 1988, pp 44-51). Oncogenes and tumor-suppressing genes have a basic distinguishing feature. The oncogenes identified thus far have arisen only in somatic cells, and thus have been incapable of transmitting their effects to the germ line of the host animal. In contrast, mutations in tumor- suppressing genes can be identified in germ line cells, and are thus transmissible to an animal's progeny.

The classic example of a hereditary cancer is retinoblastomas in children. The incidence of retinoblastomas is determined by a tumor suppressor gene, the retinoblastoma (rb or RB) gene (Weinberg, R.A. , Scientific Amer.. Sept. 1988, pp 44-51); Hansen M.F. et al. , Trends Genet. 4.:125-128 (1988); Lee, W.-H. et al.. "Molecular Biology of the Human Retinoblastoma Gene" In: Tumor Suppressor Genes. Klein, G. (ed.). Marcel Dekker, Inc., pp 169-200 (1990); all herein incorporated by reference) . Individuals born with a lesion in one of the RB alleles are predisposed to early childhood development of retinoblastomas (Vogel, F., Human Genetics 52:1-54 (1979)). Inactivation or mutation of the second RB allele in one of the somatic cells of these susceptible individuals appears to be the molecular event that leads to tumor formation (Cavenee, W.K. et al.. Nature 305:779- 784 (1983)). The RB tumor-suppressing gene has been localized onto human chromosome 13. The mutation may be readily transmitted through the germ line of afflicted individuals (Cavenee, W.K. et al.. New Engl. J. Med. 314:1201-1207 (1986)). Individuals who have mutations in only one of the two naturally present alleles of this tumor-suppressing gene are predisposed to retinoblastoma. Inactivation of the second of the two alleles is, however, required for tumorigenesis (Knudson, A.G., Proc. Nat'l. Acad. Sci. (U.S.A.) 62:820-823 (1971)). Rb mutations have been found in a large number of other human tumors, such as osteosarcoma, etc.). For a review, see Lee, E.Y.-H., "Diverse Mutations Lead to Inactivation of the Retinooblastoma Gene," In: The Molecular Biology of the Retina: Basic and Clinically Relevant Studies. Wiley-Liss, Inc., pp. 221-240 (1991), herein incorporated by reference) . A second tumor-suppressing gene is the p53 gene (Green, M.R. , Cell 56:1-3 (1989); Mowat et al.. Nature 314:633-636 (1985); Lane, P.D. et al.. Genes Devel. 4:1-8 (1990)). The protein encoded by the p53 gene is a nuclear protein that forms a stable complex with both the SV40 large T antigen and the adenovirus E1B 55 kd protein. The p53 gene product may be inactivated by binding to these proteins.

Initially, the p53 gene was thought to be an oncogene rather than a tumor-suppressing gene since it is capable of immortalizing primary rodent cells and can cooperate with the ras oncogene to cause transformation. Subsequent research revealed that the p53 genes used in those early experiments was a mutant allele of the normal p53 gene (Green, M.R., Cell 56:1-3 (1989); Lane, P.D. et al. , Genes Devel. 4_.:l-8 (1990)). Thus, the p53 gene is a tumor-suppressing gene rather than an oncogene.

Mutations at any of a large number of positions in the p53 gene can result in the activation of the transforming potential of the p53 gene product (Eliyahu et al.. Nature 312:646-649 (1984); Finlav et al.. Molec. Cell. Biol. 2:531-539 (1988)). This has suggested that the activation of the p53 transforming activity is due to the inactivation of the normal p53 activity (Green, M.R. , Cell 5_6:l-3 (1989)) . The p53 gene has been implicated as having a role in colorectal carcinoma (Baker, S.J. et al.. Science 244.:217-221 (1989)). Studies had shown that allelic deletions of the short arm of chromosome 17 occurred in over 75% of colorectal carcinomas. The region deleted was subsequently found to encompass the p53 gene locus (Baker, S.J. et al.. Science 244:217-221 (1989)). The deletion of the region was found to mark a transition from a (benign) adenocarcinoma stage to a (malignant) carcinomatous stage (Vogelstein, B. et al.. New Engl. J. Med. 319:525 (1988)).

Similar deletions in chromosome 17 have been identified in a wide variety of cancers including breast and lung cancers (Mackay, J. et al.. Lancet ii:1384

(1988); James, CD. et al.. Cane. Res. 4_8:5546 (1988);

Yakota, J. et al.. Proc. Nat'l. Acad. Sci. (U.S.A.)

24.:9252 (1987); Toguchida et al. _r Cane. Res. 48:3939 (1988)). In addition to p53 allele loss, Nigro et al. (Nature 342:705-708 (1989)) have demonstrated that the single remaining p53 allele in a variety of human tumors (brain, colon, breast, lung) undergo a point mutation which renders it tumorigenic. Fearon et al. (Cell j61:759-767 (1990)) have hypothesized that both point mutations and deletions in the p53 alleles may be required for a fully tumorigenic phenotype. Indeed, p53 mutations are the most common mutation identified in human tumors. These findings suggest that the p53 gene may have a role in many types of cancers.

VII. Conclusions

The application of the above-described technologies has the potential to produce animals which cannot be produced through classical genetics. For example, animals can be produced which suffer from, or which -are predisposed to, retinoblastoma, and other tumors associated with the loss of one or both functional alleles of the rb gene, and thus may be valuable in elucidating therapies for such diseases. Chimeric and transgenic animals have substantial use as probes of natural gene expression.

Leder, P. et al. (U.S. Patent 4,736,866) disclose the production of transgenic non-human mammals which contain cells having an exogenously added activated oncogene sequence. Although the animals are disclosed as being useful for assaying for carcinogenic materials, the precise location and structure of the added oncogene sequence in the animals is unknown, and cannot be experimentally controlled. Thus, the value of the animals as a model for oncogenesis is significantly impaired.

Despite the successes of the above-described techniques, the methods have not yet led to the development of a model transgenic animal which can be used to study the conditions responsible for the initiation of neoplasia, and which can be used as a means for developing suitable antineoplastic agents and therapies. Indeed, prior to the present invention, research on transgenic or chimeric animals containing mutations in oncogene and "critical" genes had suggested that it would not be possible to produce viable animals containing mutations in the chromosomal alleles of their tumor-suppressing genes. It was believed that such animals would be non-viable, or would not survive to maturity. See, Soriano, P. et al.. Cell 64:693-702

(1991) [c-srcl : McMahon, A. and Bradley, B. , Cell

62:1073-1085 (1990) rWnt-11 : Roller, B. et al.. Science

242:1227-1230 (1990) ΓMHC-11 : Tybulewicz, V.L.J. , Cell

.65:1153-1164 (1991) rc-abll : Mucenski, M.L. , Cell 65:677-

Animals predisposed to neoplastic disease associated with the loss of one rb allele would facilitate a better understanding of cancer; they could be used to assay for the presence of mutagenic agents in food, waste products, etc. ; they could also be used to identify agents capable of suppressing or preventing neoplastic disease. Such animals—would, therefore, be extremely desirable. The present invention provides such animals, and the methods to produce and use them. BRIEF DESCRIPTION OF THE FIGURE

Figure 1 shows the targeting strategy used to produce a defined rb mutation. A — Mouse RB Genome ("Figure 1A" in text) depicts the wild type genomic locus region exons 19-23. RB DNA fragments isolated from a mouse (129/J) genomic library (Doetschman, T.C et al. _P J. Embryo1. Exper. Morphol. 82:27-45 (1985) ) . An 8.0 kb fragment extending from a Bglll site in exon 19 -to a BamHI site in intron 22 was used for the construction of the targeting vector. B — pMG5RB-neo-2TK ("Figure IB" in text) depicts the targeting vector. The neo is PGKneo, TK are MCItk cassettes. C — Homologous Recombination ("Figure IC" in text) depicts the predicted structure of the mutated allele. Insertion of the neomycin cassette into the exon 20 disrupts the coding sequence of the RB gene. Abbreviations are E: EcoRI; B: BamHI; Bg: Bgll; H: Hindlll; K: Kpnl; N:NcoI; S: Sail; X: Xbal. The expected restriction fragments for the endogenous sequence are for Hindlll: 12.5 Kb (Probe A or Probe B) , — (Probe C) ; for Ncol: 9.5 kb (Probe A or Probe B) , — (Probe C) ; for EcoRI: — (Probe A), 10 Kb (Probe B) , and — (Probe C) . The expected restriction fragments for the recombinant sequence are for Hindlll: 7.5 Kb (Probe A or Probe C) , 6.5 Kb (Probe B) ; for Ncol: 7.0 Kb (Probe A), 4.0 Kb (Probe B) , 7.0 + 4.0 Kb (Probe C) ; for EcoRI: — (Probe A), 11.5 Kb (Probe B or Probe C).

SUMMARY OF THE INVENTION:

The present invention provides a desired non-human animal or an animal (including human) cell which contains a predefined mutation in a chromosomal allele of an rb gene that alters the espression of that gene. In one embodiment, the alteration renders the non-human animal or animal cell predisposed to tumors associated with the -15/1 - loss of a functional allele of the retinoblastoma gene. In a second embodiment, the alteration corrects a mutation in the allele that had increased the cell's neoplastic potential. The invention additionally pertains to the use of such non-human animals or animal cells, and their progeny in research and medicine.

In detail, the invention provides a transgenic or chimeric human or non-human animal cell whose genome comprises two chromosomal alleles of an rb gene, wherein at least one of the two alleles contains a predefined mutation.

-16-

The invention also includes the embodiments of the above animal cell wherein one of the alleles expresses a normal rb gene product, or wherein the cell is an embryonic stem cell. The invention also provides a non-human transgenic or chimeric animal having a human or non-human animal cell whose genome comprises two chromosomal alleles of an rb gene, wherein at least one of the two alleles contains a predefined mutation, or a progeny of the animal, or an embryonic stage ancestor of the animal.

The invention also includes the embodiments of the above non-human transgenic or chimeric animal wherein the animal cell is a germ-line cell, or a somatic cell; or wherein the animal and the animal cell are of the same or different species.

The invention also provides a non-human animal containing an embryonic stem cell of a non-human animal whose genome comprises two chromosomal alleles of an rb gene, wherein at least one of the two alleles contains a predefined mutation, or a progeny of the animal, or an embryonic stage ancestor of the animal.

The invention also provides a method for determining whether an agent is capable of affecting a characteristic of a human or non-human animal cell that is attributable to the presence or expression of an rb gene, the method comprising:

A) administering an amount of the agent to an animal cell in cell culture, the cell having a genome that comprises two chromosomal alleles of the rb gene, wherein at least one of the two alleles contains a predefined mutation;

--- B) maintaining the cell culture for a desired period of time after the administration;

C) determining whether the administration of the agent has affected a characteristic of the animal cell that is attributable to the presence or expression of the alleles of the rb gene. -17-

The invention encompasses the embodiments of the above methods wherein the agent is able to increase or able to decrease a neoplastic potential associated with the loss of a functional allele of the rb gene of the animal cell.

The invention also includes the embodiments of the above methods wherein the animal cell is an embryonic stem cell; or wherein the animal and the animal cell are of the same or different species. The invention also provides a method of gene therapy comprising altering the genome of a cell of a human or non-human animal, wherein the cell has a genome that comprises two chromosomal alleles of an rb gene, wherein at least one of the two alleles contains a mutation, to thereby form a cell wherein the mutation-containing allele has been altered such that it expresses a normal rb gene product.

DESCRIPTION OF THE PREFERRED EMBODIMENTS:

Cancer in humans develops through a multi-step process, indicating that multiple changes must occur to convert a normal cell into one with a malignant phenotype. One class of involved genes includes cellular oncogenes. When activated by mutation or when expressed inappropriately, dominant-acting oncogenes override normal cellular control mechanisms and promote unbridled cell proliferation. A newly recognized class of cellular genes that appears to be equally important in cancer development includes the tumor suppressor genes, sometimes called "anti-oncogenes." These genes act to dampen cell growth; inactivation of their normal function appears to be a common denominator in the evolution of tumor cells. The term "allele" as used herein is intended to include any nucleotide sequence of the gene that affects the expression of the particular gene. It thus is -18-

intended to encompass any enhancer, promoter, processing, intervening, coding or termination sequence or region of the gene, or any sequence that stabilizes the gene product, or its mRNA, etc. As is well known, an allele may be capable of being expressed by the natural processes operating in a cell. The expression of an allele results in the production of a gene product.

A cell's "genome" consists of all of its heritable DNA (either chromosomal or non-chromosomal (i.e., episomal, viral, etc.). As used herein, an allele is said to be "chromosomal" if it either is, or replaces, one of the two alleles of a gene which a cell inherits from its ancestors, or which an animal inherits from its parents. An allele is "non-chromosomal", as that term is used herein, if the allele increases the copy number of the total number of alleles of a particular gene which are present in a cell.

One of the two chromosomal alleles of a gene is provided by the animal's or cell's maternal parent; the other set is provided by its paternal parent. As is well known, the cells of humans and animals (especially, rodents (i.e., mouse, rat, hamster, etc.) , rabbits, sheep, goats, fish, pigs, cattle and non-human primates) are "diploid" cells, and thus naturally contain two copies ("alleles") of each and every gene of their genome. The diploid nature of human and animal cells is described by DeRobertis, E.D.P., et al. (Cell Biology. 6th Ed., W.B. Saunders Company, Philadelphia, (1975)), and in other similar treatises of cell biology. Both alleles of a tumor suppressor gene must be inactivated to result in loss of function in the cell. Inactivation of one allele increases the probability that an event will damage the surviving allele, and thus serves to make the host more susceptible to tumor induction.

The present invention relates-to the production of non-human transgenic and chimeric animals and cells which -19-

contain a "predefined" "alteration" in at least one chromosomal allele of the retinoblastoma (rb) tumor suppressor gene. The term "alteration," as that term is used herein, refers to a change in the nucleotide sequence of a gene. The alteration can cause a "mutation" that abolishes, attenuates or impairs the normal transcription, translation, expression, or processing of a gene. Alternatively, an alteration can restore, or enhance the normal transcription, transla- tion, expression, or processing of a "mutated" gene. An alteration is said to be "predefined," if the sequence that results from the alteration is (or can be) determined prior to effecting the alteration. Thus, for example, a spontaneous reversion of a mutated gene to a wild-type form is not a predefined alteration. Similarly, a naturally occuring variant gene is not a predefined alteration. Where the cells and non-human animals of the present invention contain mutations in both of their chromosomal alleles, such mutations may be the same, or they may be different from one another.

An allele of a gene is said to be mutated if (1) it is not expressed in a cell or animal, (2) the expression of the allele is altered with respect to the expression of the normal allele of the gene, or (3) the allele expresses a gene product, but that gene product has altered structure, activity, or characteristics relative to the gene product of a normal allele of that gene. -

Thus, the terms "mutation" or "mutated" as used herein are intended to denote an alteration in the "normal" or "wild-type" nucleotide sequence of any nucleotide sequence or region of the allele. As used herein, the terms "normal" and "wild-type" are intended to be synonymous, and to denote any nucleotide sequence typically found in nature, and associated with the generally encountered expression of a gene. As will be appreciated, the "wild-type" form of a gene may be a single nucleotide sequence, or may be a set of related -20-

"variant" sequences that are found in nature. The terms "mutated" and "normal" are thus defined relative to one another; where a cell has two chromosomal alleles of a gene that differ in nucleotide sequence, at least one of these alleles is a "mutant" allele as that term is used herein. A "normal tumor-suppressing gene product" is the gene product that is expressed by a "normal" tumor- suppressing gene.

A mutation may be "cryptic." A cryptic mutation does not affect either the expression of the mutated gene, or the activity or function of the expressed gene product. Cryptic mutations may be detected through nucleotide sequence analysis. Examples of cryptic mutations include mutations that do not result in a change in the amino acid sequence of the expressed gene product, as well as mutations that result in the substitution of an equivalent amino acid at a particular position in the expressed gene product. Most preferably, the mutation will be "non-cryptic" and will therefore introduce a change in the nucleotide sequence of the allele that detectably alters either the expression or the activity or function of the allele. A "mutation that detectably alters the expression of an allele," as used herein denotes any change in nucleotide sequence affecting the extent to which the allele is transcribed, translated, expressed or processed. Such alterations may be, for example, in an enhancer, promoter, coding or termination region of the allele, mutations which stabilize the gene product, or its mRNA, etc. A "mutation that detectably alters the activity of an allele," as used herein denotes any change in nucleotide sequence that alters the capacity of the expressed gene product to mediate a function of the gene product. Such mutations include changes that diminish or inactivate one or more • functions of the expressed product. Significantly, such mutations also include changes that result in an increase the capacity of the gene product to -21-

mediate any function (for example, a catalytic or binding activity) of that gene product. A "mutation that detectably alters the function of an allele," as used herein denotes any change in nucleotide sequence that alters the capacity of a binding molecule (such as a binding protein) to specifically bind to the allele.

Any of a wide variety of methods (treatment with mutagenic compounds, spontaneous isolation, insertional inactivation, site-specific insertions, deletions or substitutions, homologous recombination, etc.) may be used to produce mutations in accordance with the present invention. As indicated above, a large number of such mutations are known, and mutations can be readily identified by sequencing, tumorigenicity, resilience to tumorigenicity, binding activity, etc. (see, for example, Eliyahu et al.. Nature 312:646-649 (1984) ; Finlay et al.. Molec. Cell. Biol. 8:531-539 (1988); Nigro, J.M. et al.. Nature 342:705-708 (1989), all herein incorporated by reference) . The cells that can be produced in accordance with the present invention include both "germ-line" and "somatic" cells. A germ-line cell is a sperm cell or egg cell, or a precursor or progenitor of either; such cells have the potential of transmitting their genome (including the altered tumor-suppressor allele) in the formation of progeny animals. A somatic cell is a cell that is not a germ-line cell. Such cells may be "substantially free of naturally occurring contaminants," or may be present in an animal of the same or of different species. A cell is "substantially free of naturally occurring contaminants" when it, or an "embryonic stage" ancestor of the cell, has been purified from tissue (normal, tumor, etc.) in which the cell is, or would be, naturally associated. Two species are said to be the same if they are capable of breeding with one another to produce fertile offspring. Two species are said to be different if they are either incapable of -22-

breeding to produce viable offspring, or are substantially incapable of producing fertile offspring.

I. The rb Tumor Suppressor Gene

The present invention is directed to the formation of cells and non-human animals that contain predefined alterations in a chromosomal allele of the rb tumor suppressor gene. The rb gene is reviewed by Weinberg, R.A. (Scientific Amer.. Sept. 1988, pp 44-51); Hansen M.F. et al. (Trends Genet. 4:125-128 (1988); Lee, W.-H. et al. , "Molecular Biology of the Human Retinoblastoma Gene" In: Tumor Suppressor Genes. Klein, G. (ed.), Marcel Dekker, Inc., pp 169-200 (1990), all herein incorporated by reference.

The cDNA and genomic forms of the rb gene have been cloned (Friend, S. et al.. Nature 323:643 (1986); Lee, W. et al.. Science 235:1394 (1987); Brookstein, R. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 25:2210-2214 (1988); Fung, Y. et al.. Science 236:1657 (1987); Hong, F. et al.. Proc. Nat'l. Acad. Sci. (U.S.A.) 86_:5502 (1989); T'ang, A. et al.. Oncogene 4.:401-408 (1989), all herein incorporated by reference) . Mutations that alter the activity of the rb gene product have been described (Dunn, J.M. et al.. Molec. Cell. Biol. 9_:4596-4604 (1989); Shew, J.-Y. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 22:6-10 (1990); Bookstein, R. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 85:2210-2214 (1988); Horowitz, J.M. et al.. Science 243:937-940 (1989), all herein incorporated by reference) . Potential methods and animal (including transgenic) models for the study of retinoblastoma are discussed in Lee, W. et al.. WO 90/05180, herein incorporated by reference. The ability to manipulate this gene and to produce non-human transgenic animals which carry such mutated alleles is illustrated with respect to a particular mutated rb allele. It is to be understood, however, that the -23-

invention and the methods disclosed herein, can be used to produce any possible predefined mutation in the rb gene. In particular, the invention includes the production of animal cells and non-human transgenic or chimeric animals which carry the particular mutations of the rb gene that are associated with inactivation of the retinoblastoma gene as it occurs in retinoblastoma and other tumors associated with the loss of a functional rb allele. It would be desirable to produce a transgenic animal whose genome possesses one normal and functional rb allele and one non-functional (mutant) rb allele. Such animals could be used to study the consequences resulting from the loss of one rb allele, and thus would more clearly aid in elucidating the processes of oncogenesis and tumorigenesis. Such animals would also be useful in screening potential carcinogens, in developing novel antineoplastic therapeutics, and in gene therapy. The present invention provides such an animal.

II. Homologous Recombination

The present invention uses the process of homologous recombination to introduce a specific mutation into the naturally present rb sequence of an animal cell, most preferably an embryonic stem (ES) cell. The mutated ES cells of non-human animals can then be either cultured in suitable cell culture medium, or injected into a host embryo to make a chimeric animal and introduced into the uterus of a suitable recipient and permitted to develop into a non-human animal.

Alternatively, the methods of the present invention may be used to alter the somatic cells of a non-human animal to produce a chimeric non-human animal. An understanding of the process of homologous recombination (Watson, J.D., In: Molecular Biology of the Gene. 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA -24-

(1977) , which reference is incorporated herein by reference) is thus desirable in order to fully appreciate the present invention.

In brief, homologous recombination is a well-studied natural cellular process which results in the scission of two nucleic acid molecules having identical or substantially similar sequences (i.e., "homologous"), and the ligation of the two molecules such that one region of each initially present molecule is now ligated to a region of the other initially present molecule (Sedivy, J.M. , Bio-Technol. 6:1192-1196 (1988), which reference is incorporated herein by reference) .

The frequency of recombination between two DNA molecules may be enhanced by treating the introduced DNA with agents which stimulate recombination. Examples of such agents include trimethylpsoralen, UV light, etc.

III. Production of Chimeric and Transgenic Animals: Gene Targeting Methods

One approach to producing animals having defined and specific genetic alterations has used homologous recombination to control the site of integration of an introduced marker gene sequence in tumor cells and in fusions between diploid human fibroblast and tetraploid mouse erythroleukemia cells (Smithies, O. et al.. Nature 212:230-234 (1985)).

This approach was further exploited by Thomas, K. R. , and co-workers, who described a general method, known as "gene targeting," for targeting mutations to a preselected, desired gene sequence of an ES cell in order to produce a transgenic animal (Mansour, S.L. et al. , Nature 336:348-352 (1988); Capecchi, M.R. , Trends Genet. 5:70-76 (1989); Capecchi, M.R. , Science 244_.:1288-1292 (1989) ; Capecchi, M.R. et al.. In: Current Communications in Molecular Biology. Capecchi, M.R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), pp. 45-52; -25-

Frohman, M. A. et al.. Cell 56:145-147 (1989); all of which references are incorporated herein by reference) .

It may now be feasible to deliberately alter any gene in a mouse (Capecchi, M.R., Trends Genet. 5_:70-76 (1989); Frohman, M. A. et al.. Cell 56:145-147 (1989)). Gene targeting involves the use of standard recombinant DNA techniques to introduce a desired mutation into a cloned DNA sequence of a chosen locus. That mutation is then transferred through homologous recombination to the genome of a pluripotent, embryo-derived stem (ES) cell. The altered stem cells are microinjected into mouse blastocysts and are incorporated into the developing mouse embryo to ultimately develop into chimeric animals. In some cases, germ line cells of the chimeric animals will be derived from the genetically altered ES cells, and the mutant genotypes can be transmitted through breeding.

Gene targeting has been used to produce chimeric and transgenic mice in which an nptll gene has been inserted into the 3₂-microglobulin locus (Roller, B.H. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 26:8932-8935 (1989); Zijlstra, M. et al.. Nature 342:435-438 (1989); Zijlstra, M. et al.. Nature 344:742-746 (1989)). Similar experiments have enabled the production of chimeric and transgenic animals having a c-abl gene which has been disrupted by the insertion of an nptll gene (Schwartzberg, P.L. et al.. Science 246:799-803 (1989)). The technique has been used to produce chimeric mice in which the en-2 gene has been disrupted by the insertion of an nptll gene (Joyner, A.L. et al.. Nature 338:153-155 (1989) ) .

Gene targeting has also been used to correct an hprt deficiency in an hprf ES cell line. Cells corrected of the deficiency were used to produce chimeric animals. Significantly, all of the corrected cells exhibited gross disruption of the regions flanking the hprt locus; all of the cells tested were found to contain at least one copy -26-

of the vector used to correct the deficiency, integrated at the hprt locus (Thompson, S. et al.. Cell 56:313-321 (1989) ; Roller, B.H. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 86:8927-8931 (1989)). In order to utilize the "gene targeting" method, the gene of interest must have been previously cloned, and the intron-exon boundaries determined. The method results in the insertion of a marker gene (i.e., the nptll gene) into a translated region of a particular gene of interest. Thus, use of the gene targeting method results in the gross destruction of the gene of interest.

Recently, chimeric mice carrying the homeobox hox

1.1 allele have been produced using a modification of the gene targeting method (Zimmer, A. et al.. Nature 338:150- 154 (1989) . In this modification, the integration of vector sequences was avoided by microinjecting ES cells with linear DNA containing only a portion of the hox 1.1 allele, without any accompanying vector sequences. The DNA was found to cause the gene conversion of the cellular hox allele. Selection was not used to facilitate the recovery of the "converted" ES cells, which were identified using the polymerase chain reaction ("PCR") . Approximately 50% of cells which had been clonally purified from "converted" cells were found to contain the introduced hox 1.1 allele, suggesting to Zimmer, A. et al. either chromosomal instability or contamination of sample. Nor<e of the chimeric mice were found to be able to transmit the "converted" gene to their progeny (Zimmer, A. et al.. In: Current Communications in Molecular Biology. Capecchi, M.R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, NY (1989),-pp. 53-58).

Significantly, the use of gene targeting to alter a gene of a cell results in the formation of a gross alteration in the sequence of that gene. The efficiency of gene targeting depends upon a number of variables, and is different from construct to construct. For the rb -27-

gene constructs used herein, such efficiency is approximately 1/20, when positive negative selection is applied.

IV. Production of Chimeric and Transgenic Animals: Use of Insertion Vectors

In contrast to the above-described methods, the present invention uses a replacement vector to produce predefined alterations in the sequence of at least one of the two alleles of the rb gene of a human or animal cell. Although the methods discussed below are capable of mutating both alleles of the cell's rb gene, it is possible to readily identify (for example through the use of Southern hybridization (discussed below) , or other methods) such dual mutational events. Since the frequency of such dual mutational events is the square of the frequency of a single mutational event, cells having mutations in both of their rb alleles will be only a very small proportion of the total population of mutated cells.

The present invention has several embodiments. In the most preferred embodiment, the DNA molecule(s) which are to be introduced into the recipient cell contains a region of homology with a region of the rb gene. In a preferred embodiment, the DNA molecule will contain two regions having homology with the cell's rb gene. These "regions of homology" will preferably flank the precise sequence whose incorporation into the rb gene is desired. The DNA molecule(s) may be single stranded, but are preferably double stranded. The DNA molecule(s) may be introduced to the cell as one or more RNA molecules which may be converted to DNA by reverse transcriptase or by other means. Preferably, the DNA molecule will be a double stranded linear molecule. In the best mode for conducting the invention, such a molecule is obtained by cleaving a closed covalent circular molecule to form a -28-

linear molecule. Preferably, a restriction endonuclease capable of cleaving the molecule at a single site to produce either a blunt end or staggered end linear molecule is employed. Most preferably, the nucleotides on each side of this restriction site will comprise at least a portion of the preferred two regions of homology between the DNA molecule being introduced and the rb gene. In the construction of the altered rb genes that are described below, the Sail enzyme was used to linearize the targeting vector at the 5' end of the tk cassette.

In particular, the invention permits the replacement of the naturally present rb gene sequence of a recipient cell with an "analog" sequence. A sequence is said to be an analog of another sequence if the two sequences are substantially similar in sequence, but have minor changes in sequence corresponding to single base substitutions, deletions, or insertions with respect to one another, or if they possess minor multiple base alterations. Such alterations are intended to exclude insertions of dominant selectable marker genes.

When the predefined gene sequence, flanked by regions of homology with the rb gene sequence of the recipient cell, is introduced into the recipient cell as a linear double stranded molecule, whose termini correspond to the regions of homology, a single recombination event with the rb gene of the cell will occur in approximately 5% of the transfected cells. Such a single recombinational event will lead to the integra- tion of the entire linear molecule into the genome of the recipient cell.

The DNA molecule containing the desired gene sequence may be introduced into the pluripotent cell by any method which will permit the introduced molecule to undergo recombination at its regions of homology. Some methods, such as direct microinjection, or calcium phosphate transformation, may cause the introduced -29-

molecule to form concatemers upon integration. These concatemers may resolve themselves to form non- concatemeric integration structures. Since the presence of concatemers is not desired, methods which produce them are not preferred. In a preferred embodiment, the DNA is introduced by electroporation (Toneguzzo, F. et al.. Nucleic Acids Res, ljj:5515-5532 (1988); Quillet, A. et al. , J. Immunol. 141:17-20 (1988); Machy, P. et al. _r Proc. Natl. Acad. Sci. (U.S.A.) 85:8027-8031 (1988); all of which references are incorporated herein by reference) .

After permitting the introduction of the DNA molecule(s), the cells are cultured under conventional conditions, as are known in the art. In order to facilitate the recovery of those cells which have received the DNA molecule containing the desired gene sequence, it is preferable to introduce the DNA containing the desired gene sequence in combination with a second gene sequence which would contain a detectable marker gene sequence. For the purposes of the present invention, any gene sequence whose presence in a cell permits one to recognize and clonally isolate the cell may be employed as a detectable marker gene sequence. In one embodiment, the presence of the detectable marker sequence in a recipient cell is recognized by hybridization, by detection of radiolabelled nucleotides, or by other assays of detection which do not require the expression of the detectable marker sequence. Preferably, such sequences are detected using PCR (Mullis, K. et al.. Cold Spring Harbor Sy p. Quant. Biol. 51:263-273 (1986); Erlich H. et al.. EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K. , EP 201,184; Mullis K. et al.. US 4,683,202; Erlich, H. , US 4,582,788; and Saiki, R. et al.. US 4,683,194), which references are incorporated herein by reference) . -30-

PCR achieves the amplification of a specific nucleic acid sequence using two oligonucleotide primers complementary to regions of the sequence to be amplified. Extension products incorporating the primers then become templates for subsequent replication steps. PCR provides a method for selectively increasing the concentration of a nucleic acid molecule having a particular sequence even when that molecule has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single or double stranded DNA.

Most preferably, however, the detectable marker gene sequence will be expressed in the recipient cell, and will result in a selectable phenotype. Examples of such preferred detectable gene sequences include the hprt gene (Littlefield, J.W. , Science 145:709-710 (1964), herein incorporated by reference), the tk gene (i.e., thymidine kinase gene) and especially the tk gene of herpes simplex virus (Giphart-Gassler, M. et al.. Mutat. Res. 214:223- 232 (1989) herein incorporated by reference) , the nptll gene (Thomas, K.R. et al.. Cell 51:503-512 (1987); Mansour, S.L. et al.. Nature 336:348-352 (1988), both references herein incorporated by reference) , or other genes which confer resistance to amino acid or nucleoside analogues, or antibiotics, etc.

Cells which express an active HPRT enzyme are unable to grow in the presence of certain nucleoside analogues (such as 6-thioguanine, 8-azapurine, etc.), but are able to grow in media supplemented with HAT (hypoxanthine, aminopterin, and thymidine) . Conversely, cells which fail to express an active HPRT enzyme are unable to grow in media containing HATG, but are resistant to analogues such as 6-thioguanine, etc. (Littlefield, J.W. , Science 145:709-710 (1964)). Cells expressing active thymidine kinase are able to grow in media containing HAT, but are unable to grow in media containing nucleoside analogues such as bromo-deoxyuridine (Giphart-Gassler, M. et al. , -31-

Mutat. Res. 214:223-232 (1989)). Cells containing an active HSV-tk gene are incapable of growing in the presence of gangcylovir or similar agents.

The detectable marker gene may be any gene which can complement for a recognizable cellular deficiency. Thus, for example, the gene for HPRT could be used as the detectable marker gene sequence when employing cells lacking HPRT activity. Thus, this agent is an example of agents may be used to select mutant cells, or to "negatively select" for cells which have regained normal function.

The nptll gene (Southern, P.J., et al.. J. Molec. Appl. Genet. 1:327-341 (1982); Smithies, O. et al.. Nature 317:230-234 (1985), which references are incorporated herein by reference) is the most preferred detectable marker gene sequence. Constructs which contain both an nptll gene and either a tk gene or an hprt gene are especially preferred.

V. The -(Production of Chimeric and Transgenic

Animals

The chimeric or transgenic animal cells of the present invention are prepared by introducing one or more DNA molecules into a precursor pluripotent cell, most preferably an ES cell, or equivalent (Robertson, E.J. , In: Current Communications in Molecular Biology. Capecchi, M.R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, NY (1989), pp. 39-44, which reference is incorporated herein by reference) . The term "precursor" is intended to denote only that the pluripotent cell is a precursor to the desired ("transfected") pluripotent cell which is prepared in accordance with the teachings of the present invention. The pluripotent (precursor or transfected) cell may be cultured in_vivo, in a manner known in the art (Evans, M.J. et al.. Nature 292:154-156 (1981)) to form a chimeric or transgenic animal. The -32-

transfected cell, and the cells of the embryo that it forms upon introduction into the uterus of a female are herein referred to respectively, as "embryonic stage" ancestors of the cells and animals of the present invention.

Any ES cell may be used in accordance with the present invention. It is, however, preferred to use primary isolates of ES cells. Such isolates may be obtained directly from embryos such as the CCE cell line disclosed by Robertson, E.J., In: Current Communications in Molecular Biology. Capecchi, M.R. (ed.), Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) , pp. 39-44) , or from the clonal isolation of ES cells from the CCE cell line (Schwartzberg, P.A. et al.. Science 246:799-803 (1989) , which reference is incorporated herein by reference) . Such clonal isolation may be accomplished according to the method of E.J. Robertson (In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. (E.J. Robertson, Ed.), IRL Press, Oxford, 1987) which reference and method are incorporated herein by reference. The purpose of such clonal propagation is to obtain ES cells which have a greater efficiency for differentiating into an animal. Clonally selected ES cells are approximately 10-fold more effective in producing transgenic animals than the progenitor cell line CCE. For the purposes of the recombination methods of the present invention, clonal selection provides no advantage. An example of ES cell lines which have been clonally derived from embryos are the ES cell lines, AB1 (hprt*) or AB2.1 (hprf^}.

The ES cells are preferably cultured on stromal cells (such as STO cells (especially SNL76/7 STO cells) and/or primary embryonic G418^R fibroblast cells) as described by E.J. Robertson (In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. (E.J. Robertson, Ed.), IRL Press, Oxford, 1987, pp 71-112), which reference is incorporated herein by reference. -33-

Methods for the production and analysis of chimeric mice are disclosed by Bradley, A. (In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. (E.J. Robertson, Ed.), IRL Press, Oxford, 1987, pp 113-151), which reference is incorporated herein by reference. The stromal (and/or fibroblast) cells serve to eliminate the clonal overgrowth of abnormal ES cells. Most preferably, the cells are cultured in the presence of leukocyte inhibitory factor ("lif") (Gough, N.M. et al. , Reprod. Fertil. Dev. 1:281-288 (1989); Yamamori, Y. et al.. Science 246:1412-1416 (1989), both of which references are incorporated herein by reference) . Since the gene encoding lif has been cloned (Gough, N.M. et al. _f Reprod. Fertil. Dev. 1:281-288 (1989)), it is especially preferred to transform stromal cells with this gene, by means known in the art, and to then culture the ES cells on transformed stromal cells that secrete lif into the culture medium.

ES cell lines may be derived or isolated from any species (for example, chicken, etc.), although cells derived or isolated from mammals such as rodents (i.e., mouse, rat, hamster, etc.), rabbits, sheep, goats, fish, pigs, cattle, primates and humans are preferred.

VI. Uses of the Present Invention

The present invention provides human or animal cells which contain a desired, predefined, subtle alteration in the gene sequence of at least one of the two rb alleles of the cell's genome.

In a first embodiment, the invention also provides a means for producing non-human chimeric or transgenic animals whose cells contain such a sequence. The animals which may be produced through application of the described method include chickens, non-human mammals (especially, rodents (i.e., mouse, rat, hamster, etc.), -34-

rabbits, sheep, goats, fish, pigs, cattle and non-human primates) .

The cells and non-human animals of the present invention have both diagnostic and therapeutic utility.

A. Diagnostic Utility

Since the invention provides a cell, or a transgenic or chimeric non-human animal that contains a single functional allele of the rb gene, and since such cells will become tumor cells upon the mutation of the functional allele to a non-functional form, the present invention can be used to identify an agent affects the "neoplastic potential" of the cell or animal. As used herein, the neoplastic potential of a cell or animal is the capacity of a cell to become neoplastic, or the capacity of an animal to form tumors, due to a character¬ istic of an animal cell that is attributable to the presence or expression of a tumor-suppressing gene) . A characteristic of an animal cell is said to be "due to a characteristic of an animal cell that is attributable to the presence or expression of a tumor-suppressing gene," if the characteristic is altered by the absence or lack of expression of the tumor-suppressing gene. Examples of such characteristics include neoplastic potential associated with the loss of a functional rb allele of an animal (i.e., the frequency or predisposition of the animal to retinoblastoma or other tumors) , resilience to retinoblastoma, the extent, distribution, incidence, location, grade, etc. of retinoblastoma, etc.

In one embodiment, such agents can decrease the neoplastic potential associated with the loss of a functional rb allele of the cells or animals. Such agents are discussed below with regard to the therapeutic potential of the invention.

In a second embodiment, such agent can increase the neoplastic potential associated with the loss of a -35-

functional rb allele of the cells or animals. Thus, the cells and non-human animals of the present invention have utility in testing potential or suspected carcinogens for a role in increasing the risk that an animal will exhibit retinoblastoma. They may be used to identify and assess the effects of agents that may be present, for example, in the environment (such as environmental pollutants in air, water or soil) , or resulting from environmental exposures to chemicals, radioisotopes, etc., on the occurence (or severity, grade, etc.) of retinoblastoma or other tumor. They may also be used to facilitate studies of the effects of diet on retinoblastoma. They may be used to determine whether potential or present food additives, chemical waste products, chemical process by- products, water sources, proposed or presently used pharmaceuticals, cosmetics, etc., influence the neoplastic potential associated with the loss of a functional rb allele of of cells or animals. They may also be used to determine the influence of various energy forms (such as UV rays, X-rays, ionizing radiation, gamma rays of elemental isotopes, etc.) on the neoplastic potential associated with the loss of a functional rb allele of cells or animals.

The frequency at which a mutational event occurs is dependent upon the concentration of a mutagenic chemical agent, or the intensity of a mutagenic radiation. Thus, since the frequency of a single cell receiving two mutational events is the square of the frequency at which a single mutational event will occur, the cells and non- human animals of the present invention shall be able to identify neoplastic (mutagenic) agents at concentrations far below those needed to induce neoplastic changes in natural cells or animals.

One especially preferred cell is a non-human cell in which one of the natural rb alleles has been replaced with a functional human rb allele and the other of the natural rb alleles has been mutated to a non-functional -36-

form. Alternatively, one may employ a non-human cell in which the two natural rb alleles have been replaced with a functional and a non-functional allele of the human rb gene. Such cells may be used, in accordance with the methods described above to assess the neoplastic potential of agents in cells containing the human rb allele. More preferably, such cells are used to produce non-human animals which do not contain any natural functional rb alleles, but which contain only one functional human rb allele. Such non-human animals can be used to assess the tumorigenicity of an agent in a non-human animal expressing the human rb gene product.

1. In Vitro Assays

In one embodiment, one may employ the cells of the present invention, in in vitro cell culture, and incubate such cells in the presence of an amount of the agent whose effect on the neoplastic potential associated with the loss of a functional rb allele of a cell or animal is to be measured. This embodiment therefore comprises an in vitro assay of tumorigenic activity.

Although many carcinogenic agents may directly mediate their tumorigenic effects, some agents will not exhibit tumorigenic potential until metabolized, or until presented to a susceptible cell along with one or more "co-carcinogenic" factors. The present invention permits the identification of such "latent" carcinogenic and "co- carcinogenic" agents. In accordance with this embodiment of the invention, the presence of a "latent" carcinogen can be identified by merely maintaining cell or animal exposure to a candidate agent. Alternatively, the cells of the present invention can be incubated in "spent" culture medium (i.e;, medium containing the candidate agent that was used to culture other cells before being used to culture the cells of the present invention) . -37-

The present invention permits the identification of co-carcinogenic factors capable of inducing tumors in the presence of a second agent. Such factors can be identified by culturing the cells of the present invention in the presence of two or more candidate agents simultaneously, and then assaying for tumors.

The transformation of the cells to a neoplastic state would be indicative of tumorigenic (or neoplastic) activity of the assayed agent. Such a neoplastic state may be evidenced by a change in cellular morphology, by a loss of contact inhibition, by the acquisition of the capacity to grow in soft agar, or most preferably, by the initiation of expression of tumor antigens.

The use of tumor antigens as a means of detecting neoplastic activity is preferred since such antigens may be readily detected.

As is well known in the art, antibodies, or fragments of antibodies, may be used to quantitatively or qualitatively detect the presence in the tumor of antigens on cell surfaces. Since any cell type (i.e., lung, kidney, colon, etc.) may be employed to form the rb- mutated cells of the present invention, it is possible to determine whether an agent has a tissue specific tumorigenic potential. To accomplish this goal, one would incubate a candidate agent in the presence of rb- mutated cells derived from any of a variety of tissue types. Since tumors have tumor-specific antigens, -and since antibodies capable of binding to such antigens have been isolated, it is possible to use such antibodies to characterize any tumor antigens which may be expressed by the rb-mutated cells.

Such detection may be accomplished using any of a variety of immunoassays and radioimmune assays. A large number of different immunoassay formats have been described (Yolken, R.H., Rev. Infect. Pis. 4:35 (1982); Collins, W.P., In: Alternative Immunoassays_r John Wiley & Sons, NY (1985); Ngo, T.T. et al.. In: Enzyme Mediated -38-

Immunoassav. Plenum Press, NY (1985)). A good description of a radioimmune assay (RIA) may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S., et al.. North Holland Publishing Company, NY (1978) , with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., incorporated by reference herein.

The above-described in vitro assays have the advantageous features of potentially lower cost than presently used assays, and the capacity to readily screen large numbers of agents. Use of this embodiment facilitates comparisons of test results obtained at different times and conditions. Moreover, because it is possible to use very large numbers of cells in such assays, it is possible to detect the tumorigenic activity of tumorigenic agents even at very low concentrations. Lastly, since this embodiment can be performed using human cells, it provides a means for determining the tumorigenic (or neoplastic) potential of a test compound on human cells.

2. In Vivo Assays

In a second embodiment, one may employ the non-human animals of the present invention, and provide to such animals (by, for example, inhalation, ingestion, injection, implantation, etc.) an amount of the agent whose tumorigenic potential is to be measured. The formation of tumors in such animals (as evidenced by direct visualization by eye, or by biopsy, imaging, detection of tumor antigens, etc.) would be indicative of tumorigenic activity of the assayed agent.

The use of the non-human animals of the present invention is preferred over naturally occurring non-human animals since such natural animals contain two functional rb alleles, and thus require two mutational events in -39-

order to lead to loss of functional rb activity. In contrast, since the non-human animals of the present invention have only one functional rb allele, only one mutational event is needed to cause total loss of rb function.

The detection of tumors in such animals can be accomplished by biopsy, imaging, or by assaying the animals for the presence of cells which express tumor antigens. For example, such detection may be accomplished by removing a sample of tissue from a subject and then treating the isolated sample with any suitably labeled antibodies (or antibody fragments) as discussed above. Preferably, such in situ detection is accomplished by removing a histological specimen from the subject, and providing the labeled antibody to such specimen. The antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a sample of tissue. Through the use of such a procedure, it is possible to determine not only the presence of antigen, but also the distribution of the antigen on the examined tissue. Using the present invention, those of ordinary skill will readily perceive that any of a wide variety of histological methods (such as staining procedures) can be modified in order to achieve such in situ detection.

Alternatively, the detection of tumor cells may be accomplished by in vivo imaging techniques, in which the labeled antibodies (or fragments thereof) are provided to the subject, and the presence of the tumor is detected without the prior removal of any tissue sample. Such in vivo detection procedures have the advantage of being less invasive than other detection methods, and are, moreover, capable of detecting the presence of antigen- expressing cells in tissue which cannot be easily removed from the patient. -40-

Additionally, it is possible to assay for the presence of tumor antigens in body fluids (such as blood, lymph, etc.), stools, or cellular extracts. In such immunoassays, the antibodies (or antibody fragments) may be utilized in liquid phase or bound to a solid-phase carrier, as described below.

The use of an in vivo assay has several advantageous features. The in vivo assay permits one not only to identify tumorigenic agents, but also to assess the kind(s) of tumors induced by the agent, the number and location (i.e., whether organ or tissue specific) of any elicited tumors, and the grade (clinical significance) of such elicited tumors. It further permits an assessment of tumorigenicity which inherently considers the possible natural metabolism of the introduced agent, the possibility that the introduced agent (or its metabolic by-products) might selectively accumulate in specific tissues or organs of the recipient animal, the possibility that the recipient animal might recognize and repair or prevent tumor formation. In short, such an assay provides a true biological model for studying and evaluating the tumorigenic potential of an agent in a living non-human animal.

3. Immunoassays of Tumor Antigens

The in vitro, in situ, or in vivo detection of tumor antigens using antibodies (or fragments of antibodies) can be improved through the use of carriers. Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an -41-

antigen. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Those skilled in the art will note many other suitable carriers for binding monoclonal antibody, or will be able to ascertain the same by use of routine experimentation.

The binding molecules of the present invention may also be adapted for utilization in an immunometric assay, also known as a "two-site" or "sandwich" assay. In a typical immunometric assay, a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support that is insoluble in the fluid being tested (i.e., blood, lymph, liquified stools, tissue homogenate, etc.) and a quantity of detectably labeled soluble antibody is added to permit detection and/or quantitation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody. Typical immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody-antigen complex. After a suitable incubation period, the solid support is washed to remove the residue of the fluid sample, including unreacted antigen, if any, and then contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule") . After a second incubation period to permit the labeled antibody to complex with the antigen bound to the solid support through the unlabeled antibody, the solid support is washed a second time to remove the unreacted labeled antibody. This type of forward sandwich assay may be a simple "yes/no" assay to determine whether antigen is present or may be made quantitative by comparing the measure of labeled antibody with that obtained for a -42-

standard sample containing known quantities of antigen. Such "two-site" or "sandwich" assays are described by Wide at pages 199-206 of Radioimmune Assay Method, edited by Rirkham and Hunter, E. & S. Livingstone, Edinburgh, 1970.

In another type of "sandwich" assay, which may also be useful to identify tumor antigens, the so-called "simultaneous" and "reverse" assays are used. A simultaneous assay involves a single incubation step as the antibody bound to the solid support and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support is then determined as it would be in a conventional "forward" sandwich assay.

In the "reverse" assay, stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support is then determined as in the "simultaneous" and "forward" assays.

As explained above, the immunometric assays for antigen require that the particular binding molecule be labeled with a "reporter molecule." These reporter molecules or labels, as identified above, are conventional and well-known to the art. in the practice of the present invention, enzyme labels are a preferred embodiment. No single enzyme is ideal for use as a label in every conceivable immunometric assay. Instead, one must determine which enzyme is suitable for a particular assay system. Criteria important for the choice of -43-

enzymes are turnover number of the pure enzyme (the number of substrate molecules converted to product per enzyme site per unit of time) , purity of the enzyme preparation, sensitivity of detection of its product, ease and speed of detection of the enzyme reaction, absence of interfering factors or of enzyme-like activity in the test fluid, stability of the enzyme and its conjugate, availability and cost of the enzyme and its conjugate, and the like. Included among the enzymes used as preferred labels in the immunometric assays of the present invention are peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, glycoamylase, malate dehydrogenase, and glucose-6- phosphate dehydrogenase. Urease is among the more preferred enzyme labels, particularly because of chromogenic pH indicators which make its activity readily visible to the naked eye.

B. Therapeutic Utility

Significantly, the cells and animals of the present invention can be used to identify agents that decrease the tumorigenic (or neoplastic) potential of the cells or animals. Such agents can be "anti-tumor agents" and/or "chemopreventative agents." "Anti-tumor agents" act to decrease the proliferation of the cells (or the growth, dissemination, or metastasis of tumors in the chimeric or transgenic animals) . "Chemopreventative agents" act to inhibit the formation of new tumors. Such agents may have general activity (inhibiting all new tumor formation) , or may have a specific activity inhibiting the distribution, frequency, grade, etc.) of specific types of tumors in specific organs and tissue. Thus, the present invention permits the identification of novel antineoplastic therapeutics. Any of the above assays of tumor-suppressing activity may be used for this purpose. -44-

The transgenic cells and non-human animals of the present invention can be used to study human gene regulation of the rb gene. For example, such cells and animals can be used to investigate the interactions of the rb gene with oncogenes or other tumor suppressor genes. Thus, they may be used to identify therapeutic agents which have the ability to impair or prevent neoplastic or tumorigenic development. Such agents have utility in the treatment and cure of cancer in humans and animals.

Significantly, potential therapeutic agents are frequently found to induce toxic effects in one animal model but not in another animal model. To resolve the potential of such agents, it is often necessary to determine the metabolic patterns in various species, and to then determine the toxicities of the metabolites. The present invention permits one to produce transgenic cells or animals which could facilitate such determinations.

When providing the therapeutic agents of the present invention to the cells of an animal, pharmaceutically acceptable carriers (i.e., liposomes, etc.) are preferably employed. Such agents can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, are described, for example, in Nicolau, C et al. (Crit. Rev. Ther. Drug Carrier Svst. 2:239-271 (1989)), which reference is incorporated herein by reference.

In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the desired gene sequence together with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control the duration of action. Control release -45-

preparations may be achieved through the use of polymers to complex or absorb the desired gene sequence (either with or without any associated carrier) . The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methyl- cellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate the agent into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

C. Use in Research and in Gene Therapy

The cells and non-human animals of the present invention, quite apart from their uses in veterinary and human medicine, may be used to investigate gene regulation, expression and organization in animals. The methods of the present invention may be used to produce alterations in a regulatory region for the native rb gene sequence. Thus, the invention provides a means for altering the nature or control of transcription or translatio of the rb gene, and of altering the rb gene itself. For example, the invention enables one to introduce mutations which result in increased or -46-

decreased gene expression. Similarly, it enables one to impair or enhance the transcriptional capacity of the natural rb gene in order to decrease or increase its expression. Thus, the present invention permits the 5 manipulation and dissection of the rb gene.

Such abilities are especially valuable in gene therapy protocols, and in the development of improved animal models of retinoblastoma.

In one embodiment of the present invention, DNA

10 encoding either a functional rb gene, variants of that gene, or other genes which influence the activity of the rb gene, may be introduced into the somatic cells of an animal (particularly mammals including humans) in order to provide a treatment for cancer (i.e., "gene therapy") .

15 Most preferably, viral or retroviral vectors are employed for this purpose.

The principles of gene therapy are disclosed by Oldham, R.R. (In: Principles of Biotherapy. Raven Press, NY, 1987) , and similar texts. Disclosures of the methods

20 and uses for gene therapy are provided by Boggs, S.S. (Int. J. Cell Clon. 8:80-96 (1990)); Rarson, E.M. (Biol. Reprod. 42:39-49 (1990)); Ledley, F.D., In: Biotechnology. A Comprehensive Treatise, volume 7B. Gene Technology. VCH Publishers, Inc. NY, pp 399-458 (1989));

25 all of which references are incorporated herein by reference.

Although, as indicated above, such gene therapy can be provided to a recipient in order to treat (i.e., suppress, attenuate, or cause regression) an existing

30 neoplastic state, the principles of the present invention can be used to provide a prophylactic gene therapy to individuals who, due to inherited genetic mutations, or somatic cell mutation, contain cells having impaired rb gene expression (for example, only a single functional

35. allele of the rb gene). Such therapy would be administered in advance of the detection of cancer in -47-

order to lessen the individual's predisposition to the disease.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLE 1

INACTIVATION OF THE rb TUMOR SUPPRESSOR GENE IN THE MOUSE: OVERVIEW OF THE EXPERIMENT

Structural alterations of the rb tumor suppressor gene have been associated with a wide array of human cancers. To examine the role of rb in retinoblastoma, embryonic stems (ES) cell lines were generated in which one of the two endogenous rb alleles has been inactivated by the insertion of a neo^R gene (eg. the nptll gene of tn5) following homologous recombination.

The structure of the £b locus is depicted in Figure 1A. The gene targeting strategy utilized a murine rb genomic plasmid construct (Figure IB) that spanned rb exons 19-23. The rb sequences were flanked by an HSV TK gene and interrupted in exon 20 by a polyA - neo^R gene (the neomycin determinant) driven by a PGK promoter. The TK genes are MCItk cassettes. In the MCItk cassette, the HSV TK coding sequence is expressed from the MCI neo promoter (Stratagene) . The elements of the cassette are described by Thomas, K.R. et al. (Cell 51:503-512 (1987)). The size of the rb insert was approximately 8 kb.

Following gene transfer into the AB1 ES cells and G418/FIAU selection, clones were identified by Southern analysis which had the expected altered rb gene structure (Figure IC) . Three probes were used for this purpose. Probe C (neo probe) is used to indicate the presence of -48-

the neomycin resistance determinant in the stem cells. Probes A (part of intron 18 and exon 19) and B (part of introns 22 and 23, and exon 23) are used to reveal whether the neomycin determinant has integrated within the desired rb locus (or whether integration has been random) . If a recombinant cell contains a sequence that is capable of hybridizing with Probe C, then the cell contains the neomycin determinant. Since all cells contain the rb gene, they will all contain sequences that are capable of hybridizing with Probes A and B. However, if the neomycin determinant has integrated into the rb gene, there will be an alteration in the size of the bands detected in a Southern analysis. In this manner, it is possible to determine not only whether a particular recombinant cell contains the neomycin determinant, but also whether that determinant has integrated into the cells genome via homologous recombination.

2,210 G418^R + FIAU" clones were obtained, of which 850 were screened. These yielded 42 targeted clones, all of which appeared to be equivalent by Southern blot. 5 clones were injected into recipient C57BL/6 blastocysts, implanted into pseudopregnant C57BL/6 female mice, and permitted to develop into a chimeric or transgenic animal having a deficiency in at least one of the rb loci of its cells.

The implantation resulted in the production of 4 highly chimeric males (clones 36-8, 25-10, 16-4, and 36- 21) . These clones were found to be all the same with respect to structure of the mutated allele (except for 36-21 which does not have a disrupted RB allele) , capacity to form chimeric mice, and germ line transmission. Thus, these clones are readily formed with very good reproducibility.

The male offspring resulting from the injection of the above-described ES cell into a blastocyst were bred to generate germ line heterozygotes. Germ line transmission per male test bred for these clones was 4 -49-

OUt Of 4 (36-8); 3 out of 4 (25-10); 1 out Of 3 (16-4) and 6 out of 7 (36-21) . These heterozygotes are then examined for increased susceptibility to tumors.

EXAMPLE 2

THE GENERATION OF THE RECOMBINOGENIC rb CONSTRUCTS

To maximize an ability to obtain and select ES cells containing homologous recombination events, the strategy of Mansour et al. was utilized (Mansour, S.L. et al.. Nature 336:348-352 (1988), herein incorporated by reference) . The procedure used a positive selection method for isolating cells that had stably integrated the introduced mutating DNA and a negative selection against cells that did not contain a homologous recombination event, allowing an enrichment for cells containing the desired homologous recombination event. The adaptation of these procedures for obtaining homologous recombination into the rb gene is outlined in Figure 1. A segment of the genomic sequences from the mouse rb gene (spanning exons 19-23) was obtained from a mouse (129/J) genomic library. As shown in Figure 1 , this sequence of DNA was modified in two ways. First, a neo marker gene driven by the pgk promoter enhancer was designed to obtain high levels of expression in ES cells. This sequence was inserted into the unique Sstl site in exon 20 after being blunt-ended. This insertion provided a positive selectable marker (neo) for stable gene transfer into ES cells and disrupted the coding sequence of the rb gene, producing an inactive allele following successful homologous recombination.

The second alteration entailed the attachment of a herpes simplex virus thymidine kinase gene (HSV TK) to the 3' and 5' ends of the gene-targeting construct (Figure IB) . This attachment provided the negative selection (using the HSV-TK-specific thymidine analogue FIAU (l-(2 deoxy, 2 fluoro, 3-D arabinofuranosyl)-5- -50-

iodouracil) against cells that have random integrations of the targeting construct.

EXAMPLE 3 THE TARGETING CONSTRUCT AND

THE TRANSFECTION AND SELECTION OF ES CELLS

The murine genomic RB DNA fragment encompassing intron 18 to 23 was isolated from a 129/J genomic library (Doetschman, T.C et al.. J. Embrvol. Exper. Morphol.

22:27-45 (1985)). An 8.0 kb genomic DNA fragment

, extending from a Bglll site in exon 19 to a BamHI site in intron 22 was subcloned into a plasmid vector for the construction of the targeting vector, pMG5RB-NEO-2TK. Plasmid PGK-NEO-BPA, containing the phosphoglycerate kinase I promoter and a bovine growth hormone polyadenylation sequence (Soriano, P. et al.. Cell 24:693-702 (1991)) was used in the neomycin cassette. Briefly, an Xhol fragment was excised from the PGK-NEO- BPA plasmid, blunt-ended, and inserted into the Sstl site in exon 20 of RB genomic DNA fragment after the site was destroyed. To facilitate the construction, two Sstl sites in intron 19 and 20 and one Bglll site in intron 20 were removed by filling in with Klenow enzyme. Insertion of the neomycin cassette into the exon 20 would disrupt the coding sequence of the RB gene.

For negative selection, the plasmid, pBRTR2, containing the tk gene of herpes simplex virus under the MCI polyoma enhancer and promoter was made by cloning an EcoRI fragment (446-628) of pMCI NEOPA into an EcoRI site of pHSV-tk. To make a two-tk cassette (pBR2TK) , the BamHI site of pBRTK2 was first blunt-ended, and an Xho- Sall fragment was then subcloned into the Sail site of PBRTK2. Finally, the Bglll-BamHI fragment containing Rb genomic DNA sequences and the neomycin cassette was subcloned into the unique BamHI site of pBR2TK. The resulting construct contained two tk cassettes with -51-

opposite transcriptional orientation at both ends of the RB genomic sequences. The unique Sail site in the targeting construct, pMG-NE0-2TK, allowed the plasmid to be linearized at 5' end. The AB1 ES cell line was used as the transfection host. The cell line was derived from a Black Agouti 129 mouse (McMahon, A. and Bradley, B., Cell 62:1073-1085 (1990)). ES cells were cultured on a monolayer of mitotically inactivated mouse fibroblast SNL 76/7 STO as described (McMahon, A. and Bradley, B. , Cell 62:1073-1085 (1990)). Cells were trypsinized, resuspended at a concentration of 10⁷ per ml in PBS. Two hundreds microgram of linearized DNA were electroporated to 1 x 10⁸ cells at 230 V, 500 μF, using a Bio-Rad Gene Pulser. Cells were plated immediately after transfection, and G418 (300μg/ml, Gibco) and FIAU (0.2 μM, Bristol Myers) were added 24 hours after plating and selection were allowed to proceed for 10 to 12 days.

EXAMPLE 4

TRANSFER OF CONSTRUCTS INTO ES CELLS AND IDENTIFICATION OF CELLS CONTAINING HOMOLOGOUS RECOMBINATION EVENTS

After generating the above-described rb targeting construct (Figure IB) , the construct was introduced into cultured ES cells by electroporation. ES cells were cultured as described by E.J. Robertson (In: Teratocar¬ cinomas and Embryonic Stem Cells: A Practical Approach. (E.J. Robertson, Ed.), IRL Press, Oxford, 1987, pp 71-

112) . Cells for electroporation were collected by trypsinization at 60-80% confluency, sedimented and resuspended in buffered phosphate with DNA at 25 ng/ml.

1.7 x 10⁸ AB1 ES cells were treated with a single pulse from a Bio-Rad Gene Pulser (240 volts, 500 uF, 0.4 cm cuvette) in order to achieve electroporation. Under these conditions, efficiencies of stable gene transfer of -52-

approximately 10^"3 were obtained. After pulsing, the ES cells were plated onto SNL 76/7 feeder cells (as described above) at 10⁷ cells/90-mm plate for G418/FIAU selection. Selection was allowed to proceed for 10-12 ' days (until control plate showed no colonies) . The following results were obtained:

G418/FIAU-resistant colonies were isolated, amplified, frozen and genomic DNA purified from about 850 individual isolates. Although it had been expexted that the desired clones would be obtained only at a very low frequency, a high percentage of cells contained the desired construct.

These DNAs were restricted with Hind III and Neo I, and screened by Southern analysis using oligonucleotide probes derived from the neo gene (Probe C) and rb gene sequences outside the borders of the targeting construct (Probes A and B) . If the insertion of the construct had occurred through homologous recombination, then the following bands would be expected:

Forty two of 850 colonies tested gave the expected band pattern of a desired recombinant when analyzed by -53-

Southern hybridization. Taken together, the Southern hybridization conclusively identified colonies with rb gene disruptions generated by homologous recombination. In summary, a 17 fold enrichment was achieved to give a targeting efficiency of 1/20 per G418^R, FIAU", cell which is 2.5 x 10^"7 per cell transfected. Targeted clones were identified by Southern Analysis.

EXAMPLE 5 CONSTRUCTION OF MOUSE CHIMERAS FROM

SPECIFIC ES CELL CLONES

Chimeras were constructed from suitable clones as described by Bradley, A. (In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, (E.J. Robertson, Ed.), IRL Press, Oxford, 1987, pp 113-151), herein incorporated by reference. Briefly, 3.5-day-old blastocyst-stage embryos were collected from the dissected uterine tracts of C57BL females 3 days after plugging. 12-15 individual cells were microinjected into the cavity of the blastocyst-stage embryos and, after a brief culture period, transferred back into the uterine horns of pseudopregnant F,(CBAxC57BL) foster mothers 2 days after mating with a vasectomized male. The methods for introducing the ES cell into the blastocyst, and for producing offspring have been described above, and comprise techniques which are well- known to those of ordinary skill (Mansour, S.L. et al.. Nature 336:348-352 (1988); Capecchi, M.R. , Trends Genet. 5_:70-76 (1989); Capecchi, M.R. , Science 214_.:1288-1292 (1989) ; Capecchi, M.R. et al.. In: Current Communications in Molecular Biology. Capecchi, M.R. (ed.). Cold' Spring Harbor Press, Cold Spring Harbor, NY (1989), pp. 45-52; Frohman, M. A. et al.. Cell 56:145-147 (1989); all of which references have been incorporated herein by reference) . -54-

In order to generate an adequate number of chimeras of high quality (i.e., high contribution of donor cells) the blastocyst injection experiments were repeated over a period of a few weeks. Five clones were used to generate chimaeric mice and males from three of these clones were found to have transmitted the mutant allele through their germ line. Approximately 75 blastocyst injections were performed. The results of these injections are shown below:

The chimeras of Experiment 5 transmitted to the ES genotype, but not the mutant allele. Thus, these clones contained a mixture of targeted cells and cells that were not targeted. The non-targeted cells were transmitted at the exclusion of the pargeted cells, possibly because the targeted cells were in the minority.

EXAMPLE 6

PRODUCTION OF TRANSGENIC ANIMALS HAVING MUTANT rb ALLELES

The above-described experiments resulted in the isolation of several hundred male and female heterozygous mice having a mutant rb allele. Intercrossing heterozygous mice and the genotype the resultant litters has revealed that the Rb^'/Rb^" genotype is an embryonic lethal between 12.5 and 15.5 days of gestation. -55-

An initial inspection of the heterozygous mice for lumps caused by tumors had not revealed any spontaneous tumors in the heterozygous animals for about 6 months. However, subsequent analysis has revealed that within 11 months nearly 100% of the heterozygous animals exhibited pituitary tumors of the intermediate lobe. These tumors are fatal, and the animals die at 13-15 months.

In view of the recognition that the heterozygous animals developed intra-cranial pituitary tumors and since such tumors do not form lumps that are detectable through the gross inspection of the animals, a more detailed study of the time course of tumor development was undertaken. Thus, heterozygous mice of different ages were evaluated for the presence of pituitary tumors. This analysis revealed that pituitary tumors of the intermediate lobe could be detected in the heterozygous animals beginning at 4 months of age.

Thus, the rb heterozygous mice of the present invention have a nearly uniform predisposition to tumor formation. The mice appear morphologically normal for the first 12 months, despite the initiation of tumor formation in the intermediate lobe of their pituitary glands. The mice are able to breed progeny animals that also manifest rb heterozygosity. At 12-15 months, the pituitary tumors cause the death of the heterozygous animals.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features -56-

hereinbefore set forth and as follows in the scope of the appended claims.

Claims

-57-WHAT IS CLAIMED IS:

1. A transgenic or chimeric animal cell whose genome comprises two chromosomal alleles of an rb gene, wherein at least one of said two alleles contains a predefined mutation.

2. The animal cell of claim 1, wherein one of said alleles expresses a normal rb gene product.

3. The animal cell of claim 1 which is a human cell.

4. The animal cell of claim 1 which is a non-human animal cell.

5. The animal cell of claim 4 which is an embryonic stem cell.

6. A non-human transgenic or chimeric animal having an animal cell whose genome comprises two chromosomal alleles of an rb gene, wherein at least one of said two alleles contains a predefined mutation, or a progeny of said animal, or an embryonic stage ancestor of said animal.

7. The non-human animal of claim 6, wherein said animal cell is a human cell.

8. The non-human animal of claim 6, wherein said animal cell is a non-human cell.

9. The non-human animal of claim 8, wherein said animal cell is a germ-line cell.

10. The non-human animal of claim 8, wherein said animal cell is a somatic cell. -δδ-

ll. The non-human animal of claim 8, wherein said animal and said animal cell are of the same species.

12. The non-human animal of claim 8, wherein said animal and said animal cell are of different species.

13. A non-human animal containing the embryonic stem cell of claim 5, or a progeny of said animal, or an embryonic stage ancestor of said animal.

14. A method for determining whether an agent is capable of affecting a characteristic of an animal cell that is attributable to the presence or expression of an rb gene, said method comprising: A) administering an amount of said agent to an animal cell in cell culture, said cell having a genome that comprises two chromosomal alleles of said rb gene, wherein at least one of said two alleles contains a predefined mutation; B) maintaining said cell culture for a desired period of time after said administration;

C) determining whether the administration of said agent has affected a characteristic of said animal cell that is attributable to the presence or expression of said alleles of said rb gene.

15. The method of claim 14 wherein said agent is able to increase a neoplastic potential associated with the loss of a functional allele of the rb gene of said animal cell.

16. The method of claim 14 wherein said agent is able to decrease a neoplastic potential associated with the loss of a functional allele of the rb gene of said animal cell. -59-

17. The method of claim 14 wherein said animal cell is a human cell.

18. The method of claim 14 wherein said animal cell is _' a non-human animal cell.

19. The method of claim 18 wherein said non-human animal cell is an embryonic stem cell.

20. A method for determining whether an agent is able to affect a characteristic of an animal cell that is attributable to the presence or expression of an rb gene, said method comprising:

A) administering an amount of said agent to an animal, said animal having a cell whose genome comprises two chromosomal alleles of said £b gene, wherein at least one of said two alleles contains a predefined mutation;

B) maintaining said animal for a desired period of time after said administration; C) determining whether the administration of said agent has affected a characteristic of said cell that is attributable to the presence or expression of said alleles of said gene.

21. The method of claim 20 wherein said agent is suspected of being able to increase a neoplastic potential associated with the loss of a functional allele of the rb gene of said animal cell.

22. The method of claim 20 wherein said agent is suspected of being able to decrease a neoplastic potential associated with the loss of a functional allele of the rb gene of said animal cell.

23. The method of claim 20 wherein said animal cell is a human cell. -60-

24. The method of claim 20 wherein said animal cell is a non-human animal cell.

25. The method of claim 24 wherein said non-human animal cell is an embryonic stem cell.

26. The method of claim 20, wherein said animal and said animal cell are of the same species.

27. The method of claim 20, wherein said animal and said animal cell are of different species.

28. A method of gene therapy comprising altering the genome of a cell of an animal, wherein said cell has a genome that comprises two chromosomal alleles of an rb gene, wherein at least one of said two alleles contains a mutation, to thereby form a cell wherein said mutation- containing allele has been altered such that it expresses a normal rb gene product.

29. The method of claim 28, wherein said animal is a non-human animal.

30. The method of claim 28, wherein said animal is a human.