Quick validation of genetic quality for conditional alleles in mice

Abstract

Site‐specific conditional inactivation technologies using Cre‐loxP or Flp‐FRT systems are becoming increasingly important for the elucidation of gene function and disease mechanism in vivo. A large number of gene knockout mouse models carrying complex conditional alleles have been generated by global community efforts and made available for biomedical researchers. The structures of conditional alleles in these mice are becoming increasingly complex and sophisticated, and so the validation of the genetic quality of these alleles is likewise becoming a laborious task for individual researchers. To ensure the reproducibility of conditional experiments, the researcher should confirm that loxP or FRT is integrated at the correct positions in the genome prior to start of the experiments. We report the successful design of universal PCR primers specific to loxP and FRT for the quick validation of conditional floxed and Flrted alleles. The primer set consists of forward and reverse primers complimentary to the loxP or FRT sequences with partial modifications at the 5ʹ end containing 6‐base restriction endonuclease recognition sites. The universal primer set was tested to detect genomic intervals between a pair of cis‐integrated loxP or FRT and was useful for quickly validating various floxed or Flrted alleles in conditional mice.