Analysis of conditional gene deletion using probe based Real-Time PCR

BACKGROUND: Conditional gene deletion using Cre-lox recombination is frequently used in mouse genetics; however recombination is frequently incomplete, resulting in a mixture of cells containing the functional (2lox) allele and the truncated (1lox) allele. Conventional analysis of 1lox/2lox allele ratios using Southern Blotting is time consuming, requires relatively large amounts of DNA and has a low sensitivity. We therefore evaluated the utility of Real-Time PCR to measure 1lox/2lox allele ratios. RESULTS: We show that SYBR Green based Real-Time PCR analysis of 1lox/2lox allele ratios can generate erroneous peaks in the melting curve that are possibly caused by alternate hybridization products promoted by the palindromic loxP sequence motif. Since abnormal melting curves frequently contribute to dismissal of SYBR Green based data, we developed a convenient method with improved specificity that avoids such erroneous signals. Our data show that probe based Real-Time PCR, using a universal probe directed against the loxP site, can accurately detect small differences in 1lox/2lox allele ratios. We also validated this method in Fabpl(4× at -132)-Cre transgenic mice, measuring 1lox/2lox allele ratios that are in agreement with published data. Our Real-Time PCR protocol requires the use of one probe only for all reactions. Also the universal probe established in our assay is generally applicable to any experiment analyzing Cre-lox recombination efficiency, such that only primer sequences have to be adapted. CONCLUSIONS: Our data show that 1lox/2lox allele ratios are detected with high accuracy and high sensitivity with Real-Time PCR analysis using a probe directed against the loxP site. Due to the generally applicable probe the assay is conveniently adapted to all models of Cre-lox mediated gene deletion.