Use of Mouse Models for the Analysis of Human Disease

Christopher Semsarian1

1 Centenary Institute and University of Sydney, Australia
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 15.2
DOI:  10.1002/0471142905.hg1502s60
Online Posting Date:  January, 2009
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The first half of this unit discusses the two main approaches to developing genetically engineered mouse models of human disease (i.e., transgenesis and gene targeting). The experimental steps for each method are discussed. Potential pitfalls, advantages, and limitations are also addressed. The second half of the unit describes two mouse models of hypertrophic cardiomyopathy, which illustrate the value of studying such models of human disease, both in terms of understanding disease pathogenesis as well as potential new therapeutic avenues. Curr. Protoc. Hum. Genet. 60:15.2.1‐15.2.10. © 2009 by John Wiley & Sons, Inc.

Keywords: mouse; cardiovascular disease; transgenesis; gene targeting

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Table of Contents

  • Introduction
  • Strategic Approaches
  • Methods for Genetically Engineering Mice
  • Basic Protocol 1: DNA Purification for Transgene Microinjection
  • Basic Protocol 2: Isolation of Genomic DNA from Mouse Tails
  • Examples of Mouse Models of Human Disease
  • Summary and Future Directions
  • Literature Cited
  • Tables
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Literature Cited

   Bloch, K.D. and Grossman, B. 1995. Digestion of DNA with restriction endonucleases. Curr. Protoc. Mol. Biol. 31:3.1.1‐3.1.21.
   Brown, T. 1999. Southern blotting. Curr. Protoc. Mol. Biol. 68:2.9.1‐2.9.20.
   Budelier, K. and Schorr, J. 1998. Purification of DNA by anion‐exchange chromatography. Curr. Protoc. Mol. Biol. 42:2.1.11‐2.1.18.
   Bujard, H. 1999. Controlling genes with tetracyclines. J. Gene. Med. 1:372‐374.
   Capecchi, M.R. 1989. Altering the genome by homologous recombination. Science 244:1288‐1292.
   Chen, J., Kubalak, S.W., and Chien, K.R. 1998. Ventricular muscle‐restricted targeting of the RXRalpha gene reveals a non‐cell‐autonomous requirement in cardiac chamber morphogenesis. Dev. Ped 125:1943‐1949.
   Conner, D.A. 2000a. Mouse embryonic stem (ES) cell culture. Curr. Protoc. Mol. Biol. 51:23.3.1‐23.3.6.
   Conner, D.A. 2000b. Mouse embryonic stem (ES) cell isolation. Curr. Protoc. Mol. Biol. 52:23.4.1‐23.4.9.
   Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G.R. 2008. Immunoblotting and Immunodetection. Curr. Protoc. Mol. Biol. 83:10.8.1‐10.8.28.
   Geisterfer‐Lowrance, A.A., Christe, M., Conner, D.A., Ingwall, J.S., Schoen, F.J., Seidman, C.E., and Seidman, J.G. 1996. A mouse model of familial hypertrophic cardiomyopathy. Science 272:731‐734.
   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.
   Justice, M.J., Noveroske, J.K., Weber, J.S., Zheng, B., and Bradley, A. 1999. Mouse ENU mutagenesis. Hum. Mol. Genet. 8:1955‐1963.
   Kramer, M.F. and Coen, D.M. 2001. Enzymatic amplification of DNA by PCR: Standard procedures and optimization. Curr. Protoc. Mol. Biol. 56:15.1.1‐15.1.14.
   Lind, J.M., Chiu, C., and Semsarian, C. The genetic basis of hypertrophic cardiomyopathy. Exp. Rev. Cardiovasc. Ther. 2006. 4:927‐934.
   McConnell, B.K., Fatkin, D., Semsarian, C., Jones, K.A., Georgakopoulos, D., Maguire, C.T., Healey, M.J., Mudd, J.O., Moskowitz, I.P., Conner, D.A., Giewat, M., Wakimoto, H., Berul, C.I., Schoen, F.J., Kass, D.A., Seidman, C.E., and Seidman, J.G. 2001. Comparison of two murine models of familial hypertrophic cardiomyopathy. Circ. Res. 88:383‐389.
   Moore, D., Dowhan, D., Chory, J., and Ribaudo, R.K. 2002. Isolation and purification of large DNA restriction fragments from agarose gels. Curr. Protoc. Mol. Biol. 59:2.6.1‐2.6.12.
   Moreadith, R.W. and Radford, N.B. 1997. Gene targeting in embryonic stem cells: The new physiology and metabolism. J. Mol. Med. 75:208‐216.
   Mortensen, R. 2006. Overview of gene targeting by homologous recombination. Curr. Protoc. Mol. Biol. 76:23.1.1‐23.1.12.
   Mortensen, R. 2008a. Production of heterozygous mutant cell line by homologous recombination (single knockout). Curr. Protoc. Mol. Biol. 82:23.5.1‐23.5.11.
   Mortensen, R. 2008b. Production of homozygous mutant embryonic stem cell line (double knockout). Curr. Protoc. Mol. Biol. 82:23.6.1‐23.6.4.
   Raben, N., Lu, N., Nagaraju, K., Rivera, Y., Lee, A., Yan, B., Byrne, B., Meikle, P.J., Umapathysivam, K., Hopwood, J.J., and Plotz, P.H. 2001. Conditional tissue‐specific expression of the acid alpha‐glucosidase (GAA) gene in the GAA knockout mice: Implications for theraphy. Hum. Mol. Genet. 10:2039‐2047.
   Ramezani, A. and Hawley, R.G. 2002. Overview of the HIV‐1 Lentiviral Vector System. Curr. Protoc. Mol. Biol. 60:16.21.1‐16.21.15.
   Sanbe, A., Gulick, J., Hanks, M.C., Liang, Q., Osinska, H., and Robbins, J. 2003. Reengineering inducible cardiac‐specific transgenesis with an attenuated myosin heavy chain promoter. Circ Res. 92:609‐616.
   Tallini, Y.N., Ohkura, M., Choi, B.R., Ji, G., Imoto, K., Doran, R., Lee, J., Plan, P., Wilson, J., Xin, H.B., Sanbe, A., Gulick, J., Mathai, J., Robbins, J., Salama, G., Nakai, J., and Kotlikoff, M.I. 2006. Imaging cellular signals in the heart in vivo: Cardiac expression of the high‐signal Ca2 indicator GCaMP2. Proc. Natl. Acad. Sci. U.S.A. 103:4753‐4758.
Key References
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 2009. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
  Provides general molecular biology methods, as well as detailed protocols for generating, maintaining, and characterizing transgenic and knockout mice.
   Bedell, M.A., Jenkins, N.A., and Copeland, N.G. 1997. Mouse models of human disease. Part I: Techniques and resources for genetic analysis in mice. Genes. Dev. 11:1‐10.
  Two‐part series, providing an excellent overview of mouse genomics, with a focus on technical and practical aspects of genetic analysis in mice.
   Bedell, M.A., Largaespada, D.A., Jenkins, N.A., and Copeland, N.G. 1997. Mouse models of human disease. Part II: Recent progress and future directions. Genes. Dev. 11:11‐43.
  Describes the concept of tetracycline‐mediated regulation of gene transcription.
   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.
  Provides details of generating mouse models of human disease.
   Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. 2003. Manipulating the Mouse Embryo, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  Describes the Cre/lox system used in gene‐targeting strategies.
   Sauer, B. 1998. Inducible gene targeting in mice using the cre/lox system. Methods 14:381‐392.
Internet Resources
  Homepage of Cold Spring Harbor Laboratory.
  Homepage of Taconic Farms.
  Mouse information and services at The Jackson Laboratory.
  Transgenic and Gene Targeting Core at the University of California, San Diego Medical Center.
  Arthur Lander's Homepage, University of California, Irvine. Provides detailed and useful information on various aspects of creating genetically engineered mouse models.
  Two excellent links containing information on mouse models of human disease.
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