Overview: Engineering Transgenic Constructs and Mice

Naoto Haruyama1, Andrew Cho1, Ashok B. Kulkarni1

1 National Institute of Dental and Craniofacial Research, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 19.10
DOI:  10.1002/0471143030.cb1910s42
Online Posting Date:  March, 2009
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Cell biology research encompasses everything from single cells to whole animals. Recent discoveries concerning particular gene functions can be applied to the whole animal for understanding genotype‐phenotype relationships underlying disease mechanisms. For this reason, genetically manipulated mouse models are now considered essential to correctly understand disease processes in whole animals. This unit reviews the basic mouse technologies used to generate conventional transgenic mice, which represent a gain‐of‐function approach. First, an overview of transgenic construct design is presented. This unit then explains basic strategies for the identification and establishment of independent transgenic mouse lines, followed by comments on historical and emerging techniques. It then describes typical problems that are encountered when researchers start to generate transgenic mice. Curr. Protoc. Cell Biol. 42:19.10.1‐19.10.9. © 2009 by John Wiley & Sons, Inc.

Keywords: transgenic mice; plasmid vector; constructs; gene expression; reporter gene

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Table of Contents

  • Introduction
  • Design of the Transgenic Construct
  • Identification of Potential Founders and Genotyping Strategy
  • Establishment of Independent Mouse Lines for Experimental Use
  • General Considerations
  • Acknowledgement
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

   Brinster, R.L., Allen, J.M., Behringer, R.R., Gelinas, R.E., and Palmiter, R.D. 1988. Introns increase transcriptional efficiency in transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 85:836‐840.
   Giraldo, P. and Montoliu, L. 2001. Size matters: Use of YACs, BACs and PACs in transgenic animals. Transgenic Res. 10:83‐103.
   Goodwin, E.C. and Rottman, F.M. 1992. The 3′‐flanking sequence of the bovine growth hormone gene contains novel elements required for efficient and accurate polyadenylation. J. Biol. Chem. 267:16330‐16334.
   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. and Bujard, H. 1992. Tight control of gene expression in mammalian cells by tetracycline‐responsive promoters. Proc. Natl. Acad. Sci. U.S.A. 89:5547‐5551.
   Gu, H., Marth, J.D., Orban, P.C., Mossmann, H., and Rajewsky, K. 1994. Deletion of a DNA polymerase beta gene segment in T cells using cell type‐specific gene targeting. Science 265:103‐106.
   Huang, M.T. and Gorman, C.M. 1990. Intervening sequences increase efficiency of RNA 3′ processing and accumulation of cytoplasmic RNA. Nucleic Acids Res. 18:937‐947.
   Kozak, M. 1987. An analysis of 5′‐noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15:8125‐8148.
   Kühn, R., Schwenk, F., Aguet, M., and Rajewsky, K. 1995. Inducible gene targeting in mice. Science 269:1427‐1429.
   Lois, C., Hong, E.J., Pease, S., Brown, E.J., and Baltimore, D. 2002. Germline transmission and tissue‐specific expression of transgenes delivered by lentiviral vectors. Science 295:868‐872.
   Mayo, K.E., Warren, R., and Palmiter, R.D. 1982. The mouse metallothionein‐I gene is transcriptionally regulated by cadmium following transfection into human or mouse cells. Cell 29:99‐108.
   Rivella, S., Callegari, J.A., May, C., Tan, C.W., and Sadelain, M. 2000. The cHS4 insulator increases the probability of retroviral expression at random chromosomal integration sites. J. Virol. 74:4679‐4687.
   Rossert, J., Eberspaecher, H., and de Crombrugghe, B. 1995. Separate cis‐acting DNA elements of the mouse pro‐alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen‐producing cells in transgenic mice. J. Cell Biol. 129:1421‐1432.
   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.
   Sheets, M.D., Stephenson, P., and Wickens, M.P. 1987. Products of in vitro cleavage and polyadenylation of simian virus 40 late pre‐mRNAs. Mol. Cell Biol. 7:1518‐1529.
   Sumarsono, S.H., Wilson, T.J., Tymms, M.J., Venter, D.J., Corrick, C.M., Kola, R., Lahoud, M.H., Papas, T.S., Seth, A., and Kola, I. 1996. Down's syndrome‐like skeletal abnormalities in Ets2 transgenic mice. Nature 379:534‐537.
   Szulc, J., Wiznerowicz, M., Sauvain, M.O., Trono, D., and Aebischer, P. 2006. A versatile tool for conditional gene expression and knockdown. Nat. Methods 3:109‐116.
   Tanaka, T., Veeranna, O., Ohshima, T., Rajan, P., Amin, N.D., Cho, A., Sreenath, T., Pant, H.C., Brady, R.O., and Kulkarni, A.B. 2001. Neuronal cyclin‐dependent kinase 5 activity is critical for survival. J. Neurosci. 21:550‐558.
Key References
   Nagy, A., Gertsenstein, M., Vintersten, K., and Behringer, R. 2003. Production of transgenic mouse. In Manipulating the Mouse Embryo: A Laboratory Manual, 3rd ed. pp. 289‐358. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
  This book is one of the most read laboratory manuals for the production of transgenic mice.
   Voncken, J.W. 2003. Genetic modification of the mouse: General technology; pronuclear and blastocyst injection. In Transgenic Mouse: Methods and Protocols (M.H. Hofker and J. van Deursen, eds.) pp. 9‐34. Humana Press, Totowa, N. J.
  General mouse technologies including basic husbandry, and microinjections are very well described.
Internet Resources
  PubMed is the world's largest literature database. Search using keywords ”transgenic,” ”mouse,” and the tissue or cell type of interest to find out the preferable promoters.
  Nagy Lab Cre and Flox mouse database.
  International Mouse Strain Resource.
  Mouse genome informatics.
  These Jackson Laboratory website provide links to a variety of mouse‐related information, such as mutant resources and literature pertaining to mouse genetics.
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