PCR - Based Reverse Transcriptase Assay

ES cells. This phenomenon has been observed in our laboratory in a number of cases. Clones putatively identified as homologous recombinants by Southern analysis can be quickly checked for random insertion of extra copies of the targeting vector by PCR. Given the small difference in size between the two alleles and use of only two primers common to both WT and mutant DNA, the amplification rate of both products is similar. Therefore, any significant difference in signal intensity between the WT and floxed products indicates multiple integration of the targeting vector, which can be later confirmed by Southern analysis. The presence of multicopy insertions in CREB-loxP ES cells and HNF4γ-loxP ES clones resulted in overamplification of the mutant band (Figure 2, lanes 6 and 9). Presence of multiple copies of the mutant constructs was confirmed by Southern blot analysis (data not shown). To summarize, with the ever increasing use of the Cre-loxP system studying genetic mutations in the mouse, we present a generally applicable PCR-based approach for genotyping genomic DNA harboring loxP sites. Unlike previously reported knock-out detection methods, where PCR analysis for genotyping requires use of three primers, only two primers are required here, allowing for robust PCR assays to be set up rapidly. Due to the use of identical primers in the detection of both WT and loxP alleles, the method also allows for accurate detection of copy number differences when screening ES cells.