Genomic Analysis of Transgenic Animals
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.
KeywordsGentle Agitation Transfer Buffer Slot Blot Denaturation Buffer Size Restriction Fragment
- 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