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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Abstract

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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Materials

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Figures

  •  FigureFigure 19.10.1 Representative transgenic construct and primer designs for genotyping. (A) Typical transgene construct is depicted, consisting of promoter, intron, protein-coding sequence (cDNA) from a gene of interest, stop codon, poly(A) sequence, and enhancer sequences. The promoter sequence normally contains the transcriptional start site, although there are variations. The inclusion of an intron leads to a greater percentage of active transgene expression in mouse lines. The protein coding sequence (cDNA) needs to have a translation start site, typically consisting of a Kozak consensus sequence in addition to an ATG start codon. The entire fragment can be cut out from the vector backbone by a single restriction enzyme (site A) prior to zygote injection. A restriction enzyme site that can be cut only once inside of the fragment should be considered at the construct-design stage for future genotyping by Southern blotting. Primer set 1, designed at the junction of 2 different elements, can amplify a unique sequence. (B) Alternative strategy of primer design. The primers for genotyping can also be designed in cDNA to bridge between two or more different exons so that the endogenous gene would be amplified with a larger size at low efficiency. ATG, translation start codon; stop, translation stop codon; seq, sequence.
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  •  FigureFigure 19.10.2 Diagrams of transgenes integrated in the mouse genome and the strategy for genotyping by Southern blotting. (A) The scheme assumes there are six copies of a transgene integrated head-to-tail. (B) Example for genotyping of transgenic mouse by Southern blotting. Enzyme sites A and B in panel (A) represent hypothetical locations of the restriction enzyme sites in integrated locus. If the genomic DNA containing the transgenes is digested with enzyme A, five copies of transgenes with the size of the injection fragment (transgene) and two flanking sequences (F1 and F2) will be released from the genomic DNA. The five copies of transgene fragments will be detected as the most intense band by using a probe made from the entire injected fragment, probe 1 (transgene in panel B, lane A). The flanking fragments of the transgene, which consist of the genomic sequence and a part of the transgene, can also be detected as two obvious bands with unique sizes depending on the integration sites (bands F1 and F2 in panel B, lane A). These bands can be used as single copy standards to calculate the absolute copy numbers of the transgene. Because the transgene is randomly integrated in the mouse genome, different lines show different sizes of flanking sequences (bands F1 and F2 in panel B, lane B). The band from an endogenous promoter may also be detected in all the samples (endogenous promoter in panel B). Lane C in panel B shows a transgene band with less intensity (~1 copy), suggesting that this line may be a mosaic mouse. If more than three different flanking sequences appear, the founder mouse may have multichromosomal integration (bands F in panel B, lane E). Lane D represents the wild-type mouse control. If the transgene fragment is too long to resolve using standard Southern blotting (>15 to 20 kb), a restriction enzyme that cuts two or more times in the injected fragment can be used (enzyme site B in panel A). In this case, probe 2 detects six copies of transgene fragments generated by enzyme B with no flanking sequences.
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Literature Cited

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
    http://www.ncbi.nlm.nih.gov/sites/entrez?db=PubMed

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.

    http://www.mshri.on.ca/nagy/

Nagy Lab Cre and Flox mouse database.

    http://www.informatics.jax.org/imsr/index.jsp

International Mouse Strain Resource.

    http://www.informatics.jax.org/

Mouse genome informatics.

    http://www.jax.org/

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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