Constructing TRPC7 Overexpression and Knockdown Plasmids

Mouse TRPC7 cDNA [16 (link)] was kindly provided by Professor Yasuo Mori (Kyoto University). For constructing TRPC7 overexpression plasmid Blue-pAdTrack-CMV-TRPC7, restriction endonuclease sites KpnI and NotI were added to 5′- and 3′- end of mTRPC7 cDNA, respectively, by PCR and the cDNA was ligated into the adenoviral shuttle plasmid Blue-pAdTrack-CMV which was modified from pAdTrack-CMV plasmid (Addgene plasmid # 16405). For constructing Tag-TRPC7 overexpression plasmid Blue-pAdTrack-CMV-TagTRPC7 KpnI site plus 3× Flag was added to the 5′- end, 3× hemagglutinin (HA) plus NotI site was added to the 3′- end of mTRPC7 cDNA, the cDNA was then ligated into Blue-pAdTrack-CMV. For constructing TRPC7 knockdown plasmids pAdTrack-U6-shRNA458 and pAdTrack-U6-shRNA459, two sequences of shRNA, shRNA-458 (targeting mouse TRPC7) and shRNA-459 (targeting rat TRPC7), adopted from Genetic Perturbation Platform were synthesized and ligated into the adenoviral shuttle plasmid pAdTrack-U6 at AgeI and XhoI restriction sites. pAdTrack-U6 was modified from pAdTrack plasmid (Addgene plasmid # 16404). The plasmid pAdTrack-U6-shRNAluc harboring shRNA targeting luciferase (shRNA-luc) was constructed as a negative control [31 (link)]. Targeting sequences of these shRNAs were shRNA-458: 5′-GCCGAATCAAACTCGCCATTA-3′, shRNA-459: 5′-GCCAACATTGAGACTGAATTT-3′, shRNA-luc: 5′-CCTAAGGTTAAGTCGCCCTCG-3′.