Failure to amplify templates containing the synthetic loxP3 region using flanking primers.

<p><b>A.</b> Primers targeting Wt sequences upstream (F^Flank1) and downstream (R^Flank1) of the synthetic loxP3 region were predicted to amplify a PCR product of 453 bp from the KO^1st 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>B.</b> PCR with these primers is predicted to produce 453 bp and 386 bp products from KO^1st chimeras and a single 386 bp product from Wt samples. <b>C.</b> A single product is amplified from KO^1st chimeras and Wt mice samples using the F^Flank1 with R^Flank1 primers. PCR was performed with an annealing temperature of 56°C and products were separated on a 1% agarose gel containing GelRed stain. <b>D.</b> Sanger sequencing of the PCR products from C confirms that only amplicons containing the Wt-specific identifier were produced. <b>E.</b> PCR with F^loxP3 and R^Flank1 primers using DNA isolated from four F1 progeny (derived from crossing male and female KO^1st chimeras) identified two individuals as positive for the KO^1st template. <b>F</b>. PCR with F^Flank1 with R^Flank1 primers failed to distinguish the KO^1st 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 KO^1st lanes in C represent PCR products derived from the same two distinct KO^1st chimeric mice presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165012#pone.0165012.g001" target="_blank">Fig 1</a>. The Wt and KO^1st chimera DNA samples in F are isolated from brain tissue; these samples are also used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165012#pone.0165012.g003" target="_blank">Fig 3</a>. <i>Abbreviations</i>: KO^1st: Knockout first chimera, F1: Filial hybrid 1, P: Primers only.