It is absolutely essential to develop a robust screening assay that can reliably distinguish between a rare targeted recombination event and the far more frequent random integration of the targeting vector. It is also critical to determine recombination at both the 5' and 3' arm. Depending on the length of the arms, long-range PCR, Southern blot analysis or a combination of both can be used for the initial screen. Irrespective of the method, it is important that the strategy include a probe and at least one primer positioned outside the region of homology. For PCR-based screening, the second primer can be designed to a vector-specific element such as the neo cassette or a loxP site. Targeted recombination at one arm does not necessarily mean that recombination occurred at the other arm and therefore, independent screening strategies must be developed for each arm.
When designing a screening strategy it is easiest, whenever possible, to develop a PCR-based screen for the shortest arm that can be used to initially analyze clones. This will most likely substantially reduce the number of clones that will need to be screened by Southern for recombination at the remaining arm.
It is, however, important to develop probes for both arms as confirmation of thawed clones by Southern blot is recommended prior to blastocyst injection.
We recommend establishing screening conditions initially using high quality genomic DNA. While probe specificity and sensitivity can be determined on wild-type DNA, testing PCR primers presents a greater challenge, as the paired primer set will only work following targeted recombination. Therefore, test primer sets can be designed that test the functionality of each individual primer on wild-type genomic DNA. Once assay parameters have been established, primers/probes should be tested with DNA isolated directly from 96-well plates. (Protocol)