A simple qPCR-based method to detect correct insertion of homologous targeting vectors in murine ES cells

The identification of correctly targeted embryonic stem (ES) cell clones from among the large number of random integrants that result from most selection paradigms remains an important hurdle in the generation of animals bearing homologously targeted transgenes. Given the limitations inherent to Southern blotting and standard PCR, we utilized quantitative real-time polymerase chain reaction (qPCR) to rapidly identify murine ES cell clones containing insertions at the correct genomic locus. Importantly, this approach is useful for screening ES clones from conditional/insertional "knock-in" strategies in which there is no loss of genetic material. Simple validation avoids the generation of assays prone to false negative results. In this method, probe and primer sets that span an insertion site detect and quantify the unperturbed gene relative to an irrelevant reference gene, allowing ES cell clones to be screened for loss of detection of one copy of the gene (functional loss of homozygousity (LOH)) that occurs when the normal DNA is disrupted by the insertion event. Simply stated, detected gene copy number falls from two to one in correctly targeted clones. We have utilized such easily designed and validated qPCR LOH assays to rapidly and accurately identify insertions in multiple target sites (including the Lepr and mTOR loci) in murine ES cells, in order to generate transgenic animals.