Identification of the linkage of the K14-Agouti cassette to the MMTV-Cre transgene using inverse PCR.

<p>(A) The schematic diagram of the inverse PCR. (B) PCR using specific primer sets identified the K14-agouti cassette adjacent to the MMTV-Cre transgene. The linkage of the linkage of the MMTV-Cre and K14-agouti transgenes allow distinction of the line A, line D and line F founder lines. The MMTV-Cre cassette <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019233#pone.0019233-Freeburn1" target="_blank">[5]</a> also contains a rabbit growth hormone intron sequence (RGI). The K14-Agouti cassette <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019233#pone.0019233-Kucera1" target="_blank">[14]</a> also contains a human growth hormone polyadenylation sequence (GHA). The Cre-F/Cre-R primer set identifies the presence of Cre gene. The K14P-3R/GHA-1F primer set identifies the presence of the K14-agouti cassette. The MMTV-1R/GHA-1R primer set identifies the presence of the head-to-tail linkage of the K14-agouti cassette and the MMTV-Cre cassette. The RGI-1F/GHA-1R primer set identifies the presence of the tail-to-tail linkage of the MMTV-Cre cassette and the K14-agouti cassette. 1, wild type DNA; 2, MMTV-Cre DNA used in this study; 3, MMTV-Cre line A DNA from Jackson lab; 4, MMTV-Cre line D from Jackson lab; 5, MMTV-Cre line A DNA from Hennighausen lab; 6, MMTV-Cre line F DNA from Hennighausen lab; N, no DNA control. Note, lane 2, 3, and 5 (all Line A) have the same PCR fragments for the head-tail and the tail-tail linkages whereas lane 4 (line D), and lane 6 (line F) only have PCR fragments for the tail-tail linkage. The sizes of PCR products are listed under each DNA gel panel.