Genomic Analysis of Transgenic Animals

Southern Blotting
  • Duncan R. Smith
  • David Murphy
Part of the Methods in Molecular Biology™ book series (MIMB, volume 18)

Abstract

Although subsequent generations can be rapidly screened using slot blotting (Chapter 35) or the polymerase chain reaction (PCR; Chapter 36), founder transgenic animals should initially be analyzed by Southern blotting for two reasons. First, low copy number transgenics or mosaic transgenics (where only a small number of the cells in a biopsy may be transgenic, and hence the effective copy number of the transgene in the DNA preparation is less than one) may not be clearly identifiable using slot blots because of high background. Such animals can, however, be unambiguously identified by the presence of bands of predicted sizes on Southern blot filters (1,2). Note that PCR can also clearly detect low copy-number transgenes. Second, both slot blot and PCR are unable to give direct information on transgene copy number, transgene integrity (i.e., the presence or absence of gross deletions, insertions, and so forth), and the number of independent transgene integration sites. Careful choice of restriction enzymes used to cleave the DNA, followed by Southern blotting, can reveal information about all of these features of a transgenic animal.

Keywords

Glycerol EDTA Agarose Bromide Electrophoresis 

References

  1. 1.
    Murphy, D. and Hanson, J. (1987) The production of transgenic mice by the microinjection of cloned DNA into fertilized one-cell eggs, in DNA Cloning, A Practical Approach, vol. 3 (Glover, D. M., ed.), IRL Press, Oxford, UK, pp. 213–248.Google Scholar
  2. 2.
    Southern, E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1993

Authors and Affiliations

  • Duncan R. Smith
    • 1
  • David Murphy
    • 2
  1. 1.Molecular Neurobiology Laboratory, Institute of Molecular and Cell BiologyNational University of SingaporeRepublic of Singapore
  2. 2.Neuropeptide Laboratory, Institute of Molecular and Cell BiologyNational University of SingaporeRepublic of Singapore

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