Tg(Myh6-mCherry,-Trpv4)1Td

The Tg(alpha-MHC-loxP-mCherrySTOP-loxP-TRPV4)1td transgenic construct was created using cDNA of mouse Trpv4, the pmCherryC1 vector, the pJFRC172 vector, and the vector containing the alpha-myosin heavy chain promoter. First, pmCherryC1 was digested using BglII-BamHI for removal of the multiple cloning site, followed by AgeI-MluI (with Klenow fragment) to excise the mCherry coding region and stop codons. The mCherrySTOP sequence was then subcloned into pJFRC172 (AgeI -SpeI with Klenow fragment) resulting in a floxed mCherrySTOP sequence. The loxP-mCherrySTOP-loxP coding sequence was amplified with PCR primers encoding SalI (forward) and MluI and SalI (reverse) restriction sites, and subcloned into the SalI site of the alpha-MHC vector to form alpha-MHC-loxP-mCherrySTOP-loxP. The Trpv4 coding sequence was then amplified from cDNA with forward and reverse primers encoding BssHII restriction sites, and was inserted into the MluI site of alpha-MHC-loxP-mCherrySTOP-loxP to form alpha-MHC-loxP-mCherrySTOP-loxP-TRPV4. (J:268386)