Figure 1.

Generation of the smooth muscle-specific knock out mouse model. (A) Schematic illustrating strategy for TRPC3 gene deletion. Open boxes are untranslated exon sequence. Closed boxes are translated open reading frame. Asterisks denote stop codons. Red arrows mark the location of loxP sites targeting the pore-forming region (exon 7) for cleavage by Cre recombinase. Blue arrows denote forward and reverse primers for RT-PCR analysis. (B) RT-PCR analysis of mRNA from endothelium-denuded mouse mesenteric arteries from tamoxifen-treated (TRPC3smcKO) or vehicle-treated (littermate control) mice. 500 bp floxed amplicon of exon 7 denotes an intact exon 7 while 300 bp band indicates the cleavage fragment of exon 7 excision. No cleavage fragments were found in any tissue of untreated mice whereas only the 300 bp cleavage fragment was detected in mesenteric artery of tamoxifen-treated mice, demonstrating effective knockout of TRPC3 in SMCs. (C) Brain sections fluorescently immunolabeled against TRPC3 (purple) and smooth muscle alpha actin (green). Note the co-localization (white) of TRPC3 and smooth muscle alpha actin in small cerebral arteries of a littermate control and the absence of colocalization in cerebral arteries of a TRPC3smcKO mouse.