<i>Slc7a5-</i>Flox gene construct and mouse genotyping scheme.

<p>(A) Graphical representation of the <i>Slc7a5</i>-Flox construct used to generate the chimera. Primer locations used for vector assembly and genotyping are indicated and primer sequences shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089547#pone-0089547-t001" target="_blank">Table 1</a>. LoxP sites (grey-filled triangles), FRT sites (white-filled triangles), phosphoglucokinase (PGK) promoter-driven neomycin-resistance gene (PGK-neo) and thymidine kinase gene (TK) are shown. The splice donor (SD) was incorporated into the targeting construct to allow correct splicing of the transcript generated from the PGK promoter only if the targeting construct is inserted within an intron (see B). (B) to (D) Representative PCR genotyping results. (B) RT-PCR screen of RNA from mouse embryonic stem cells using 11–14 primer pair, a 500 bp product would indicate correct joining of the exonic sequence from the splice donor with exon3 of <i>Slc7a5</i>. Gel shows RT-PCR for a positive ES cell population (+) or control cells (–). (C) Genotyping PCR for the 5′ loxP site using DNA from C57Bl/6 (WT) or a mixed litter of wild-type (2) and heterozygous Slc7a5 Flox^Neo (+/−) (3, 5, 6, 7) mice as a template resulted in either a product of 202 bp or products of 202 and 250 bp, respectively using the 9–10 primer pair. For 3′ loxP site genotyping, PCR using primer pair 12–13 (note primer 12 is duplicated in SD) generated a 230 bp product from wild-type samples or products of 230 and 318 bp from heterozygotes. (D) PCR analysis of genomic DNA from a single mixed litter of heterozygous <i>Slc7a5</i>-Flox (2,3,4,7) and <i>Slc7a5+/−</i> (1,5,6,8) mice using the 9–13 primer pair which generates a product of 235 bp only with the recombined <i>Slc7a5</i> gene lacking the 1855 bp floxed region (which includes exon1).