Gene targeting strategy to produce the <i>MCT1</i> knockout/ß-galactosidase knockin mouse.

<p>(<b>A</b>) Structure of the <i>MCT1</i> wildtype allele, the targeting vector, and the resulting <i>MCT1</i> knockout/ß-galactosidase knockin allele showing the targeted locus. Exons are represented as grey boxes except exon 1 that contains the translation initiation codon (ATG). The <i>mct1</i> gene sequence (640 bp) replaced by the fused LacZ/Neo gene sequence is indicated in red. TK, thymidine kinase gene for negative selection with Gancyclovir ; ß-Gal, the LacZ gene sequence coding for the enzyme ß-galactosidase ; Neo, neomycin resistance gene for positive selection with G418. (<b>B</b>) Southern blot performed with a 900 bp probe (green in A) on selected embryonic cell (ES) DNA digested with NdeI (Nd). ES 1 and 2 cell populations are showing a 7200 nt band corresponding to the wildtype (WT) allele and a 8800 nt band corresponding to the modified knockin (KI) allele due to a double recombination event. ES 3 and 4 cell populations did not undergo the double recombination event and only exhibit the 7200 nt band. (<b>C</b>) PCR genotyping strategy. PCR made with the primers MCT1 Fo, MCT1 Re and ßGal Re (see localization in A) present in the same reaction produces a fragment of 538 nt for wildtype (WT) animals, but also an additional fragment of 422 nt for heterozygote (HE) animals. (<b>D</b>) Histochemical detection of ß-galactosidase activity in selected tissues of <i>MCT1</i>^+/LacZ mice. The blue color in cells reflects the presence of the ß-galactosidase enzyme and therefore the <i>mct1</i> promoter activity (arrows). Tissue counterstaining was done with nuclear fast red dye. EDL, <i>extensor digitorum longus</i>.