The need for additional genotyping of ES clones obtained from KOMP, EUCOMM and NORCOMM consortia

As a rule, the structure of the targeted loci in these commercially available clones have not been confirmed by Southern and therefore it is often not clear if they contain undetected deletions anywhere along the vector. In addition, usually only the floxed exon has been verified by genomic sequencing but not any other exon that may be part of the vector. In some cases the loxP sites have also not been verified by genomic sequencing despite the fact that they can easily get lost during integration of the vector. For this reason, KOMP, EUCOMM and NORCOMM consortia strongly recommend to perform Southern blotting to confirm the integrity of the targeted allele along its entire length. In our experience between 1 in 4 and 1 in 6 clones obtained from these sources show significant deletions/abberations by Southern. Based on the above, we recommend both Southern genotyping for these ES clones, and genomic PCR to confirm the presence of both loxP sites after gene targeting. At the same time, confirmation of the lox P sites usually allows easy distinction between homozygotic and heterozygotic clones.