RRC-TPS: Transgenic Screening
Southern blot analysis of tail DNA (5 or 10ug) should be used to identify founder transgenic mice produced by pronuclear injection. For Southern analysis one needs a restriction digest that gives a unique size band(s) for the transgene, or a probe that does not hybridize with endogenous mouse genomic sequences. Select an enzyme that will produce bands of predictable size. When choosing a restriction enzyme, keep in mind that in most cases the injected DNA fragment will integrate as a head-to-tail concatemer, or array, containing a few or several copies of the fragment. You might chose an enzyme that cuts the fragment used for microinjection 1x and use the fragment, itself, as the hybridization probe. Under these conditions, one will observe either (i) a "fragment-length" band, the intensity of which will depend on the number of copies of the fragment integrated into the genome, and two bands of unpredictable size that represent the junction fragments between the integrated transgene concatemer and flanking chromosomal DNA; or (ii) two bands of unpredictable size, representing the junction fragments between a single integrated transgene and flanking DNA.
Quantitation standards should also be used. For these standards use varying amounts of the fragment used for microinjection, mixed with digested normal mouse carrier tail DNA. For initial screening, amounts of fragment equivalent to 0, 1, 5, and 10 copies per genome (DNA content is about 6 pg/genome) are good values to start with. Remember to convert these numbers to those representing the number of copies that would be present in the 5ug or 10ug of genomic DNA run in the gel. See the TPS Director for help with these calculations. Warning - it is essential to demonstrate prior to initiation of your project that your Southern blot can detect your transgene down to the single-copy level. That is, you must show that your assay will pick up animals carrying only one copy of the trangene. Please bring these preliminary results to the facility ahead of time. Note that the apparent copy number reflects both the actual copy number per genome and the fraction of cells containing the foreign DNA. Thus, if a founder mouse is "mosaic" and some cells lack the transgene (i.e., transgene integration occurred some time after the first embryonic cell division) the copy number will be underestimated. For this reason, and in the case that there are two integration sites that will segregate in the next generation, copy number should be more rigorously determined in F1 transgenic progeny.
PCR may also be used for transgenic screening. It is essential to show that your PCR assay is able to detect down to the single-copy level; this can be done, as discussed for Southern blots, by adding the calculated amount of your transgene to normal mouse carrier tail DNA. To demonstrate that tail DNA preparations are free of PCR inhibitors you should also develop a second assay that will detect an endogenous mouse gene, such as beta-globin. Keep in mind that Southerns will still be useful for determination of transgene copy-number and for revealing possible integration at two different loci in a given founder. In the latter case the two integration sites will usually differ in transgene copy-number and will generate distinct junction fragments. This means that the two integration loci will be seen to segregate among F1 offspring when analyzed by Southern blot, but willl not be revealed by PCR. For these reasons you may want to consider PCR only for routine screening of mice once you get a transgenic line going.