Failure to Genotype: A Cautionary Note on an Elusive loxP Sequence.

ABSTRACT: Here we report on a technical difficulty we encountered while optimizing genotyping strategies to identify mice derived from Exoc3l2tm1a(KOMP)Wtsi embryonic stem cells obtained from the Knockout Mouse Project Repository. The Exoc3l2tm1a(KOMP)Wtsi construct encodes a "knockout-first" design with loxP sites that confer conditional potential (KO1st). We designed primers that targeted wild-type sequences flanking the most downstream element of the construct, an 80 base pair synthetic loxP region, which BLAST alignment analysis reveals is an element common to over 10,000 conditional gene-targeting mouse models. As PCR products amplified from KO1st and wild-type templates would have different lengths (and different mobility in an agarose gel) this strategy was designed to determine the zygosity

SUBMITTER: Kreuger J 

PROVIDER: S-EPMC5074582 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature