Generation of STRA6 KO mice.

<p><i>A. Targeting strategy for generating STRA6 KO mice.</i> The red squares on the 5′ and 3′ sides of the mouse STRA6 WT genomic sequence represent the sequences serving as probes for genotyping by Southern blotting. <i>B. Genotyping of STRA6 mutant mice.</i> Tail DNA was digested with XbaI, and analyzed by Southern blotting (top panel), with the 5′ probe whose location is indicated in A. A 7.8-kb band representing the WT allele and a 5.2-kb band representing the recombinant allele are indicated by arrows. Similarly, tail DNA was digested with ScaI, and analyzed with the 3′ probe (bottom panel). A 6.9-kb band representing the WT allele and a 4.3-kb band representing the recombinant allele are indicated by arrows. <i>C. Absence of STRA6 mRNA expression in STRA6 KO splenocytes.</i> STRA6 mRNA levels from WT and KO splenocytes were analyzed by RT-qPCR. The RT-qPCR samples were in triplicate and the results are expressed as ratios of STRA6 versus β-actin mRNA signals with means ± SD indicated. The experiments were conducted twice and representative data of one experiment are shown. <i>D. Absence of STRA6 protein expression in KO thymocytes.</i> WT and KO thymocytes were stained with goat anti-mouse STRA6 Ab, and analyzed by flow cytometry. The shaded area is the isotypic Ab staining control using WT thymocytes. The thick line represents WT thymocytes stained with anti-STRA6 Ab, and the dotted line, KO thymocytes stained with anti-STRA6 Ab. The experiments were carried out three times and a representative histogram is shown.