Developing Genetically Engineered Mouse Models to Study Tumor Suppression

Shunbin Xiong1, Jan Parker‐Thornburg2, Guillermina Lozano1

1 Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas, 2 Department of Biochemistry and Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, Texas
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo110159
Online Posting Date:  March, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Since the late 1980s, the tools to generate mice with deletions of tumor suppressors have made it possible to study such deletions in the context of a whole animal. Deletion of some tumor suppressors results in viable mice while deletion of others yield embryo lethal phenotypes, cementing the concept that genes that often go awry in cancer are also of developmental importance. More sophisticated mouse models were subsequently developed to delete a gene in a specific cell type at a specific time point. Additionally, incorporation of point mutations in a specific gene as observed in human tumors has also revealed their contributions to tumorigenesis. On the other hand, some models never develop cancer unless combined with other deletions, suggesting a modifying role in tumorigenesis. This review will describe the technical aspects of generating these mice and provide examples of the outcomes obtained from alterations of different tumor suppressors. Curr. Protoc. Mouse Biol. 2:9‐24 © 2012 by John Wiley & Sons, Inc.

Keywords: knockout; knock‐in; translocation; transgenic mouse; phenotype analysis

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Gene Deletion via Knockout Targeting Strategies
  • Characterizing Tumor Models
  • Generation of Mice with Deletions of Tumor Suppressors
  • Generating Conditional Loss‐of‐Function Alleles
  • Conditional Loss‐of‐Function Alleles for Tumor Suppressors Bypass Lethality and Expand Tumor Phenotypes
  • Generating Knock‐In Alleles
  • Translocations
  • Transgenic Mice
  • Allele Preservation
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Armstrong, J.F., Kaufman, M.H., Harrison, D.J., and Clarke, A.R. 1995. High‐frequency developmental abnormalities in p53‐deficient mice. Curr. Biol. 5:931‐936.
   Arumugam, P.I., Urbinati, F., Velu, C.S., Higashimoto, T., Grimes, H.L., and Malik, P. 2009. The 3′ region of the chicken hypersensitive site‐4 insulator has properties similar to its core and is required for full insulator activity. PLoS One 4:e6995.
   Beard, C., Hochedlinger, K., Plath, K., Wutz, A., and Jaenisch, R. 2006. Efficient method to generate single‐copy transgenic mice by site‐specific integration in embryonic stem cells. Genesis 44:23‐28.
   Bigner, S.H., Mark, J., Burger, P.C., Mahaley, M.S. Jr., Bullard, D.E., Muhlbaier, L.H., and Bigner, D.D. 1988. Specific chromosomal abnormalities in malignant human gliomas. Cancer Res. 48:405‐411.
   Brinster, R.L., Chen, H.Y., Trumbauer, M., Senear, A.W., Warren, R., and Palmiter, R.D. 1981. Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs. Cell 27:223‐231.
   Brinster, R.L., Chen, H.Y., Warren, R., Sarthy, A., and Palmiter, R.D. 1982. Regulation of metallothionein‐thymidine kinase fusion plasmids injected into mouse eggs. Nature 296:39‐42.
   Cairns, P., Okami, K., Halachmi, S., Halachmi, N., Esteller, M., Herman, J.G., Jen, J., Isaacs, W.B., Bova, G.S., and Sidransky, D. 1997. Frequent inactivation of PTEN/MMAC1 in primary prostate cancer. Cancer Res. 57:4997‐5000.
   Cairns, P., Evron, E., Okami, K., Halachmi, N., Esteller, M., Herman, J.G., Bose, S., Wang, S.I., Parsons, R., and Sidransky, D. 1998. Point mutation and homozygous deletion of PTEN/MMAC1 in primary bladder cancers. Oncogene 16:3215‐3218.
   Capecchi, M.R. 1989. Altering the genome by homologous recombination. Science 244:1288‐1292.
   Chen, D., Livne‐bar, I., Vanderluit, J.L., Slack, R.S., Agochiya, M., and Bremner, R. 2004. Cell‐specific effects of RB or RB/p107 loss on retinal development implicate an intrinsically death‐resistant cell‐of‐origin in retinoblastoma. Cancer Cell 5:539‐551.
   Christophorou, M.A., Martin‐Zanca, D., Soucek, L., Lawlor, E.R., Brown‐Swigart, L., Verschuren, E.W., and Evan, G.I. 2005. Temporal dissection of p53 function in vitro and in vivo. Nat. Genet. 37:718‐726.
   Ciavatta, D., Kalantry, S., Magnuson, T., and Smithies, O. 2006. A DNA insulator prevents repression of a targeted X‐linked transgene but not its random or imprinted X inactivation. Proc. Natl. Acad. Sci. U.S.A. 103:9958‐9963.
   Clarke, A.R., Maandag, E.R., van Roon, M., van der Lugt, N.M., van der Valk, M., Hooper, M.L., Berns, A., and te Riele, H. 1992. Requirement for a functional Rb‐1 gene in murine development. Nature 359:328‐330.
   Clarke, A.R., Purdie, C.A., Harrison, D.J., Morris, R.G., Bird, C.C., Hooper, M.L., and Wyllie, A.H. 1993. Thymocyte apoptosis induced by p53‐dependent and independent pathways. Nature 362:849‐852.
   Collins, E.C., Pannell, R., Simpson, E.M., Forster, A., and Rabbitts, T.H. 2000. Inter‐chromosomal recombination of Mll and Af9 genes mediated by cre‐loxP in mouse development. EMBO Rep. 1:127‐132.
   Costantini, F. and Lacy, E. 1981. Introduction of a rabbit beta‐globin gene into the mouse germ line. Nature 294:92‐94.
   Cui, X., Ji, D., Fisher, D.A., Wu, Y., Briner, D.M., and Weinstein, E.J. 2011. Targeted integration in rat and mouse embryos with zinc‐finger nucleases. Nat. Biotechnol. 29:64‐67.
   Danielian, P.S., Muccino, D., Rowitch, D.H., Michael, S.K., and McMahon, A.P. 1998. Modification of gene activity in mouse embryos in utero by a tamoxifen‐inducible form of Cre recombinase. Curr. Biol. 8:1323‐1326.
   Derksen, P.W., Liu, X., Saridin, F., van der Gulden, H., Zevenhoven, J., Evers, B., van Beijnum, J.R., Griffioen, A.W., Vink, J., Krimpenfort, P., Peterse, J.L., Cardiff, R.D., Berns, A., and Jonkers, J. 2006. Somatic inactivation of E‐cadherin and p53 in mice leads to metastatic lobular mammary carcinoma through induction of anoikis resistance and angiogenesis. Cancer Cell 10:437‐449.
   Di Cristofano, A., Pesce, B., Cordon‐Cardo, C., and Pandolfi, P.P. 1998. Pten is essential for embryonic development and tumour suppression. Nat. Genet. 19:348‐355.
   Doetschman, T., Gregg, R.G., Maeda, N., Hooper, M.L., Melton, D.W., Thompson, S., and Smithies, O. 1987. Targeted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330:576‐578.
   Donehower, L.A. and Lozano, G. 2009. 20 years studying p53 functions in genetically engineered mice. Nat. Rev. Cancer 9:831‐841.
   Donehower, L.A., Harvey, M., Slagle, B.L., McArthur, M.J., Montgomery, C.A. Jr., Butel, J.S., and Bradley, A. 1992. Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356:215‐221.
   Donehower, L.A., Harvey, M., Vogel, H., McArthur, M.J., Montgomery, C.A. Jr., Park, S.H., Thompson, T., Ford, R.J., and Bradley, A. 1995. Effects of genetic background on tumorigenesis in p53‐deficient mice. Mol. Carcinog. 14:16‐22.
   Evans, S.C., Viswanathan, M., Grier, J.D., Narayana, M., El‐Naggar, A.K., and Lozano, G. 2001. An alternatively spliced HDM2 product increases p53 activity by inhibiting HDM2. Oncogene 20:4041‐4049.
   Friend, S.H., Bernards, R., Rogelj, S., Weinberg, R.A., Rapaport, J.M., Albert, D.M., and Dryja, T.P. 1986. A human DNA segment with properties of the gene that predisposes to retinoblastoma and osteosarcoma. Nature 323:643‐646.
   Giraldo, P. and Montoliu, L. 2001. Size matters: Use of YACs, BACs and PACs in transgenic animals. Transgenic Res. 10:83‐103.
   Gordon, J.W., Scangos, G.A., Plotkin, D.J., Barbosa, J.A., and Ruddle, F.H. 1980. Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. U.S.A. 77:7380‐7384.
   Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., and Bujard, H. 1995. Transcriptional activation by tetracyclines in mammalian cells. Science 268:1766‐1769.
   Guo, C., Yang, W., and Lobe, C.G. 2002. A Cre recombinase transgene with mosaic, widespread tamoxifen‐inducible action. Genesis 32:8‐18.
   Halachmi, N., Halachmi, S., Evron, E., Cairns, P., Okami, K., Saji, M., Westra, W.H., Zeiger, M.A., Jen, J., and Sidransky, D. 1998. Somatic mutations of the PTEN tumor suppressor gene in sporadic follicular thyroid tumors. Genes Chromosomes Cancer 23:239‐243.
   Harbour, J.W., Lai, S.L., Whang‐Peng, J., Gazdar, A.F., Minna, J.D., and Kaye, F.J. 1988. Abnormalities in structure and expression of the human retinoblastoma gene in SCLC. Science 241:353‐357.
   Hasty, P., Rivera‐Perez, J., and Bradley, A. 1991. The length of homology required for gene targeting in embryonic stem cells. Mol. Cell. Biol. 11:5586‐5591.
   Hasty, P., Rivera‐Perez, J., and Bradley, A. 1992. The role and fate of DNA ends for homologous recombination in embryonic stem cells. Mol. Cell. Biol. 12:2464‐2474.
   Hensel, C.H., Hsieh, C.L., Gazdar, A.F., Johnson, B.E., Sakaguchi, A.Y., Naylor, S.L., Lee, W.H., and Lee, E.Y. 1990. Altered structure and expression of the human retinoblastoma susceptibility gene in small cell lung cancer. Cancer Res. 50:3067‐3072.
   Hu, N., Gutsmann, A., Herbert, D.C., Bradley, A., Lee, W.H., and Lee, E.Y. 1994. Heterozygous Rb‐1 delta 20/+mice are predisposed to tumors of the pituitary gland with a nearly complete penetrance. Oncogene 9:1021‐1027.
   Hu, W., Feng, Z., Teresky, A.K., and Levine, A.J. 2007. p53 regulates maternal reproduction through LIF. Nature 450:721‐724.
   Iwakuma, T. and Lozano, G. 2007. Crippling p53 activities via knock‐in mutations in mouse models. Oncogene 26:2177‐2184.
   Jacks, T., Fazeli, A., Schmitt, E.M., Bronson, R.T., Goodell, M.A., and Weinberg, R.A. 1992. Effects of an Rb mutation in the mouse. Nature 359:295‐300.
   Jacks, T., Remington, L., Williams, B.O., Schmitt, E.M., Halachmi, S., Bronson, R.T., and Weinberg, R.A. 1994. Tumor spectrum analysis in p53‐mutant mice. Curr. Biol. 4:1‐7.
   Jones, S.N., Hancock, A.R., Vogel, H., Donehower, L.A., and Bradley, A. 1998. Overexpression of Mdm2 in mice reveals a p53‐independent role for Mdm2 in tumorigenesis. Proc. Natl. Acad. Sci. U.S.A. 95:15608‐15612.
   Jonkers, J., Meuwissen, R., van der Gulden, H., Peterse, H., van der Valk, M., and Berns, A. 2001. Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nat. Genet. 29:418‐425.
   Joyner, A.L. 2001. Gene Targeting: A Practical Approach. Oxford University Press, New York.
   Kistner, A., Gossen, M., Zimmermann, F., Jerecic, J., Ullmer, C., Lubbert, H., and Bujard, H. 1996. Doxycycline‐mediated quantitative and tissue‐specific control of gene expression in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 93:10933‐10938.
   Knudson, A.G. Jr. 1971. Mutation and cancer: Statistical study of retinoblastoma. Proc. Natl. Acad. Sci. U.S.A. 68:820‐823.
   Knudson, A.G. Jr., Meadows, A.T., Nichols, W.W., and Hill, R. 1976. Chromosomal deletion and retinoblastoma. N. Engl. J. Med. 295:1120‐1123.
   Koch, J.G., Gu, X., Han, Y., El‐Naggar, A.K., Olson, M.V., Medina, D., Jerry, D.J., Blackburn, A.C., Peltz, G., Amos, C.I., and Lozano, G. 2007. Mammary tumor modifiers in BALB/cJ mice heterozygous for p53. Mamm. Genome 18:300‐309.
   Koller, B.H., Hagemann, L.J., Doetschman, T., Hagaman, J.R., Huang, S., Williams, P.J., First, N.L., Maeda, N., and Smithies, O. 1989. Germ‐line transmission of a planned alteration made in a hypoxanthine phosphoribosyltransferase gene by homologous recombination in embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 86:8927‐8931.
   Lang, G.A., Iwakuma, T., Suh, Y.A., Liu, G., Rao, V.A., Parant, J.M., Valentin‐Vega, Y.A., Terzian, T., Caldwell, L.C., Strong, L.C., El‐Naggar, A.K., and Lozano, G. 2004. Gain of function of a p53 hot spot mutation in a mouse model of Li‐Fraumeni syndrome. Cell 119:861‐872.
   Lee, E.Y., To, H., Shew, J.Y., Bookstein, R., Scully, P., and Lee, W.H. 1988. Inactivation of the retinoblastoma susceptibility gene in human breast cancers. Science 241:218‐221.
   Lee, E.Y., Chang, C.Y., Hu, N., Wang, Y.C., Lai, C.C., Herrup, K., Lee, W.H., and Bradley, A. 1992. Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature 359:288‐294.
   Lee, G. and Saito, I. 1998. Role of nucleotide sequences of loxP spacer region in Cre‐mediated recombination. Gene 216:55‐65.
   Lee, W.H., Bookstein, R., Hong, F., Young, L.J., Shew, J.Y., and Lee, E.Y. 1987. Human retinoblastoma susceptibility gene: Cloning, identification, and sequence. Science 235:1394‐1399.
   Lei, Q., Jiao, J., Xin, L., Chang, C.J., Wang, S., Gao, J., Gleave, M.E., Witte, O.N., Liu, X., and Wu, H. 2006. NKX3.1 stabilizes p53, inhibits AKT activation, and blocks prostate cancer initiation caused by PTEN loss. Cancer Cell 9:376‐378.
   Leibo, S.P. 1986. Cryobiology: Preservation of mammalian embryos. In Genetic Engineering of Animals (J.W. Evans and A. Hollaender, eds.) pp 251‐272. Plenum Publishing Corp., New York.
   Lengner, C.J., Steinman, H.A., Gagnon, J., Smith, T.W., Henderson, J.E., Kream, B.E., Stein, G.S., Lian, J.B., and Jones, S.N. 2006. Osteoblast differentiation and skeletal development are regulated by Mdm2‐p53 signaling. J. Cell Biol. 172:909‐921.
   Li, D.M. and Sun, H. 1997. TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta. Cancer Res. 57:2124‐2129.
   Li, J., Yen, C., Liaw, D., Podsypanina, K., Bose, S., Wang, S.I., Puc, J., Miliaresis, C., Rodgers, L., McCombie, R., Bigner, S.H., Giovanella, B.C., Ittmann, M., Tycko, B., Hibshoosh, H., Wigler, M.H., and Parsons, R. 1997. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275:1943‐1947.
   Liu, G., McDonnell, T.J., Montes de Oca Luna, R., Kapoor, M., Mims, B., EI‐Naggar, A.K., and Lozano, G. 2000. High metastatic potential in mice inheriting a targeted p53 missense mutation. Proc. Natl. Acad. Sci. U.S.A. 97:4174‐4179.
   Liu, X., Holstege, H., van der Gulden, H., Treur‐Mulder, M., Zevenhoven, J., Velds, A., Kerkhoven, R.M., van Vliet, M.H., Wessels, L.F., Peterse, J.L., Berns, A., and Jonkers, J. 2007. Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1‐mutated basal‐like breast cancer. Proc. Natl. Acad. Sci. U.S.A. 104:12111‐12116.
   Lobe, C.G., Koop, K.E., Kreppner, W., Lomeli, H., Gertsenstein, M., and Nagy, A. 1999. Z/AP, a double reporter for cre‐mediated recombination. Dev. Biol. 208:281‐292.
   MacPherson, D., Sage, J., Kim, T., Ho, D., McLaughlin, M.E., and Jacks, T. 2004. Cell type‐specific effects of Rb deletion in the murine retina. Genes Dev. 18:1681‐1694.
   Mansour, S.L., Thomas, K.R., and Capecchi, M.R. 1988. Disruption of the proto‐oncogene int‐2 in mouse embryo‐derived stem cells: A general strategy for targeting mutations to non‐selectable genes. Nature 336:348‐352.
   Marine, J.C., Francoz, S., Maetens, M., Wahl, G., Toledo, F., and Lozano, G. 2006. Keeping p53 in check: Essential and synergistic functions of Mdm2 and Mdm4. Cell Death Differ. 13:927‐934.
   Meyers, E.N., Lewandoski, M., and Martin, G.R. 1998. An Fgf8 mutant allelic series generated by Cre‐ and Flp‐mediated recombination. Nat. Genet. 18:136‐141.
   Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. 2003. Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition). Cold Spring Harbor Laboratory Press, Cold Spring, Harbor, NY.
   Nakagata, N. 1995. Studies on cryopreservation of embryos and gametes in mice. Exp. Anim. 44:1‐8.
   Nakao, K., Nakagata, N., and Katsuki, M. 1998. Production of chimeric mice from cryopreserved blastocysts. Exp. Anim. 47:167‐171.
   Olive, K.P., Tuveson, D.A., Ruhe, Z.C., Yin, B., Willis, N.A., Bronson, R.T., Crowley, D., and Jacks, T. 2004. Mutant p53 gain of function in two mouse models of Li‐Fraumeni syndrome. Cell 119:847‐860.
   Ostermeier, G.C., Wiles, M.V., Farley, J.S., and Taft, R.A. 2008. Conserving, distributing and managing genetically modified mouse lines by sperm cryopreservation. PLoS One 3:e2792.
   Palmiter, R.D. and Brinster, R.L. 1986. Germ‐line transformation of mice. Annu. Rev. Genet. 20:465‐499.
   Papaioannou, V. and Johnson, R. 2001. Production of chimeras by blastocyst and morula injection of targeted ES cells. In Gene Targeting: A Practival Approach (A.L. Joyner, ed.) pp133‐175. Oxford University Press, New York.
   Pease, S. and Saunders, T.L (eds.) 2011. Advanced Protocols for Animal Transgenesis. Springer, Dordrecht, The Netherlands.
   Podsypanina, K., Ellenson, L.H., Nemes, A., Gu, J., Tamura, M., Yamada, K.M., Cordon‐Cardo, C., Catoretti, G., Fisher, P.E., and Parsons, R. 1999. Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems. Proc. Natl. Acad. Sci. U.S.A. 96:1563‐1568.
   Rincon‐Arano, H., Furlan‐Magaril, M., and Recillas‐Targa, F. 2007. Protection against telomeric position effects by the chicken cHS4 beta‐globin insulator. Proc. Natl. Acad. Sci. U.S.A. 104:14044‐14049.
   Rodda, S.J. and McMahon, A.P. 2006. Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors. Development 133:3231‐3244.
   Rodriguez, C.I., Buchholz, F., Galloway, J., Sequerra, R., Kasper, J., Ayala, R., Stewart, A.F., and Dymecki, S.M. 2000. High‐efficiency deleter mice show that FLPe is an alternative to Cre‐loxP. Nat. Genet. 25:139‐140.
   Sah, V.P., Attardi, L.D., Mulligan, G.J., Williams, B.O., Bronson, R.T., and Jacks, T. 1995. A subset of p53‐deficient embryos exhibit exencephaly. Nat. Genet. 10:175‐180.
   Salomon, B., Maury, S., Loubiere, L., Caruso, M., Onclercq, R., and Klatzmann, D. 1995. A truncated herpes simplex virus thymidine kinase phosphorylates thymidine and nucleoside analogs and does not cause sterility in transgenic mice. Mol. Cell. Biol. 15:5322‐5328.
   Sauer, B. and Henderson, N. 1988. Site‐specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc. Natl. Acad. Sci. U.S.A. 85:5166‐5170.
   Saunders, T.L. 2011. Inducible transgenic mouse models. Methods Mol. Biol. 693:103‐115.
   Schaft, J., Ashery‐Padan, R., van der Hoeven, F., Gruss, P., and Stewart, A.F. 2001. Efficient FLP recombination in mouse ES cells and oocytes. Genesis 31:6‐10.
   Shawlot, W., Siciliano, M.J., Stallings, R.L., and Overbeek, P.A. 1989. Insertional inactivation of the downless gene in a family of transgenic mice. Mol. Biol. Med. 6:299‐307.
   Smith, A.J., De Sousa, M.A., Kwabi‐Addo, B., Heppell‐Parton, A., Impey, H., and Rabbitts, P. 1995. A site‐directed chromosomal translocation induced in embryonic stem cells by Cre‐loxP recombination. Nat. Genet. 9:376‐385.
   Soriano, P. 1999. Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet. 21:70‐71.
   Stambolic, V., Tsao, M.S., Macpherson, D., Suzuki, A., Chapman, W.B., and Mak, T.W. 2000. High incidence of breast and endometrial neoplasia resembling human Cowden syndrome in pten ± mice. Cancer Res. 60:3605‐3611.
   Steck, P.A., Pershouse, M.A., Jasser, S.A., Yung, W.K., Lin, H., Ligon, A.H., Langford, L.A., Baumgard, M.L., Hattier, T., Davis, T., Frye, C., Hu, R., Swedlund, B., Teng, D.H., and Tavtigian, S.V. 1997. Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat. Genet. 15:356‐362.
   Stratton, M.R., Campbell, P.J., and Futreal, P.A. 2009. The cancer genome. Nature 458:719‐724.
   Suzuki, A., de la Pompa, J.L., Stambolic, V., Elia, A.J., Sasaki, T., del Barco Barrantes, I., Ho, A., Wakeham, A., Itie, A., Khoo, W., Fukumoto, M., and Mak, T.W. 1998. High cancer susceptibility and embryonic lethality associated with mutation of the PTEN tumor suppressor gene in mice. Curr. Biol. 8:1169‐1178.
   Sztein, J.M., Farley, J.S., and Mobraaten, L.E. 2000. In vitro fertilization with cryopreserved inbred mouse sperm. Biol. Reprod. 63:1774‐1780.
   T'Ang, A., Varley, J.M., Chakraborty, S., Murphree, A.L., and Fung, Y.K. 1988. Structural rearrangement of the retinoblastoma gene in human breast carcinoma. Science 242:263‐266.
   Tashiro, H., Blazes, M.S., Wu, R., Cho, K.R., Bose, S., Wang, S.I., Li, J., Parsons, R., and Ellenson, L.H. 1997. Mutations in PTEN are frequent in endometrial carcinoma but rare in other common gynecological malignancies. Cancer Res. 57:3935‐3940.
   te Riele, H., Maandag, E.R., and Berns, A. 1992. Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc. Natl. Acad. Sci. U.S.A. 89:5128‐5132.
   Tesson, L., Cozzi, J., Menoret, S., Remy, S., Usal, C., Fraichard, A., and Anegon, I. 2005. Transgenic modifications of the rat genome. Transgenic Res. 14:531‐546.
   Thomas, K.R. and Capecchi, M.R. 1987. Site‐directed mutagenesis by gene targeting in mouse embryo‐derived stem cells. Cell 51:503‐512.
   Tong, C., Huang, G., Ashton, C., Li, P., and Ying, Q.L. 2011. Generating gene knockout rats by homologous recombination in embryonic stem cells. Nat. Protoc. 6:827‐844.
   Tsukada, T., Tomooka, Y., Takai, S., Ueda, Y., Nishikawa, S., Yagi, T., Tokunaga, T., Takeda, N., Suda, Y., Abe, S., Matsuo, L., Ikawa, Y., and Aizawa, S. 1993. Enhanced proliferative potential in culture of cells from p53‐deficient mice. Oncogene 8:3313‐3322.
   Utomo, A.R., Nikitin, A.Y., and Lee, W.H. 1999. Temporal, spatial, and cell type‐specific control of Cre‐mediated DNA recombination in transgenic mice. Nat. Biotechnol. 17:1091‐1096.
   Van Deursen, J., Fornerod, M., Van Rees, B., and Grosveld, G. 1995. Cre‐mediated site‐specific translocation between nonhomologous mouse chromosomes. Proc. Natl. Acad. Sci. U.S.A. 92:7376‐7380.
   Venkatachalam, S., Shi, Y.P., Jones, S.N., Vogel, H., Bradley, A., Pinkel, D., and Donehower, L.A. 1998. Retention of wild‐type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation. EMBO J. 17:4657‐4667.
   Vooijs, M. and Berns, A. 1999. Developmental defects and tumor predisposition in Rb mutant mice. Oncogene 18:5293‐5303.
   Walkley, C.R., Qudsi, R., Sankaran, V.G., Perry, J.A., Gostissa, M., Roth, S.I., Rodda, S.J., Snay, E., Dunning, P., Fahey, F.H., Alt, F.W., McMahon, A.P., and Orkin, S.H. 2008. Conditional mouse osteosarcoma, dependent on p53 loss and potentiated by loss of Rb, mimics the human disease. Genes Dev. 22:1662‐1676.
   Walters, M.C., Fiering, S., Bouhassira, E.E., Scalzo, D., Goeke, S., Magis, W., Garrick, D., Whitelaw, E., and Martin, D.I. 1999. The chicken beta‐globin 5′HS4 boundary element blocks enhancer‐mediated suppression of silencing. Mol. Cell. Biol. 19:3714‐3726.
   Wang, Y., Suh, Y.A., Fuller, M.Y., Jackson, J.G., Xiong, S., Terzian, T., Quintas‐Cardama, A., Bankson, J.A., El‐Naggar, A.K., and Lozano, G. 2011. Restoring expression of wild‐type p53 suppresses tumor growth but does not cause tumor regression in mice with a p53 missense mutation. J. Clin. Invest. 121:893‐904.
   Whittingham, D.G., Leibo, S.P., and Mazur, P. 1972. Survival of mouse embryos frozen to −196 degrees and −269 degrees C. Science 178:411‐414.
   Williams, B.O., Remington, L., Albert, D.M., Mukai, S., Bronson, R.T., and Jacks, T. 1994. Cooperative tumorigenic effects of germline mutations in Rb and p53. Nat. Genet. 7:480‐484.
   Woychik, R.P. and Alagramam, K. 1998. Insertional mutagenesis in transgenic mice generated by the pronuclear microinjection procedure. Int. J. Dev. Biol. 42:1009‐1017.
   Woychik, R.P., Stewart, T.A., Davis, L.G., D'Eustachio, P., and Leder, P. 1985. An inherited limb deformity created by insertional mutagenesis in a transgenic mouse. Nature 318:36‐40.
   Wu, H., Liu, X., and Jaenisch, R. 1994. Double replacement: Strategy for efficient introduction of subtle mutations into the murine Col1a‐1 gene by homologous recombination in embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 91:2819‐2823.
   Wu, L., de Bruin, A., Saavedra, H.I., Starovic, M., Trimboli, A., Yang, Y., Opavska, J., Wilson, P., Thompson, J.C., Ostrowski, M.C., Rosol, T.J., Woollett, L.A., Weinstein, M., Cross, J.C., Robinson, M.L., and Leone, G. 2003. Extra‐embryonic function of Rb is essential for embryonic development and viability. Nature 421:942‐947.
   Xiong, S., Pant, V., Suh, Y.A., Van Pelt, C.S., Wang, Y., Valentin‐Vega, Y.A., Post, S.M., and Lozano, G. 2010. Spontaneous tumorigenesis in mice overexpressing the p53‐negative regulator Mdm4. Cancer Res. 70:7148‐7154.
   Yao, S., Osborne, C.S., Bharadwaj, R.R., Pasceri, P., Sukonnik, T., Pannell, D., Recillas‐Targa, F., West, A.G., and Ellis, J. 2003. Retrovirus silencer blocking by the cHS4 insulator is CTCF independent. Nucleic Acids Res. 31:5317‐5323.
   Yenofsky, R.L., Fine, M., and Pellow, J.W. 1990. A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. Proc. Natl. Acad. Sci. U.S.A. 87:3435‐3439.
   Zhang, H., Hasty, P., and Bradley, A. 1994. Targeting frequency for deletion vectors in embryonic stem cells. Mol. Cell. Biol. 14:2404‐2410.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library