Failure to amplify templates containing the synthetic loxP3 region using flanking primers.
A. Primers targeting Wt sequences upstream (FFlank1) and downstream (RFlank1) of the synthetic loxP3 region were predicted to amplify a PCR product of 453 bp from the KO1st construct and 386 bp from Wt sequences. The 80 bp synthetic loxP3 region replaces eleven Wt-specific bps; therefore, this represents a unique identifier for amplicons derived from Wt alleles. B. PCR with these primers is predicted to produce 453 bp and 386 bp products from KO1st chimeras and a single 386 bp product from Wt samples. C. A single product is amplified from KO1st chimeras and Wt mice samples using the FFlank1 with RFlank1 primers. PCR was performed with an annealing temperature of 56°C and products were separated on a 1% agarose gel containing GelRed stain. D. Sanger sequencing of the PCR products from C confirms that only amplicons containing the Wt-specific identifier were produced. E. PCR with FloxP3 and RFlank1 primers using DNA isolated from four F1 progeny (derived from crossing male and female KO1st chimeras) identified two individuals as positive for the KO1st template. F. PCR with FFlank1 with RFlank1 primers failed to distinguish the KO1st positive F1 progeny from Wt samples. PCR was performed with an annealing temperature of 60°C and products were separated on a 2.5% agarose gel containing GelRed stain. The two KO1st lanes in C represent PCR products derived from the same two distinct KO1st chimeric mice presented in Fig 1. The Wt and KO1st chimera DNA samples in F are isolated from brain tissue; these samples are also used in Fig 3. Abbreviations: KO1st: Knockout first chimera, F1: Filial hybrid 1, P: Primers only.