This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the paper and its Supporting Information files.įunding: This study was funded by grants awarded to JK from the Cancer Foundation (Cancerfonden: grant number CAN 2014/820 and the Swedish Research Council (Vetenskapsrådet: grant number 2010-3968. Received: JAccepted: OctoPublished: October 21, 2016Ĭopyright: © 2016 Kreuger, O’Callaghan. Anderson, University of Iowa, UNITED STATES This brief report aims to raise awareness regarding this potential source of false-negative genotype results, particularly for those who are devising genotyping strategies for similarly engineered animal models.Ĭitation: Kreuger J, O’Callaghan P (2016) Failure to Genotype: A Cautionary Note on an Elusive loxP Sequence. We discuss the possibility that secondary DNA structures, formed due to the palindromic nature of the synthetic loxP region, may have caused the KO 1st template to elude the PCR when using primers that flanked this region. Unexpectedly, while the latter strategy detected the synthetic loxP region and correctly genotyped KO 1st chimeric mice, the same individuals were genotyped as wild-type when using the primers that flanked the synthetic loxP region. In parallel we performed PCR with a primer specifically targeting the synthetic loxP sequence. As PCR products amplified from KO 1st and wild-type templates would have different lengths (and different mobility in an agarose gel) this strategy was designed to determine the zygosity of individual mice from a single PCR. 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. The Exoc3l2 tm1a( KOMP) Wtsi construct encodes a “knockout-first” design with loxP sites that confer conditional potential (KO 1st). Here we report on a technical difficulty we encountered while optimizing genotyping strategies to identify mice derived from Exoc3l2 tm1a(KOMP)Wtsi embryonic stem cells obtained from the Knockout Mouse Project Repository.
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