¦Â3ª²AR gene cloning from human bladder detrusor smooth muscle and construction of its antisense eukaryotic expression vector

    LI Xin, WANG Ping, LI Gang, LIU Yiª²Li

    Department of Urology, Fourth Affiliated Hospital, China Medical University, Shenyang 110032, China

    ¡¾Abstract¡¿ AIM: To clone human betaª²3 adrenoceptor (¦Â3ª²AR) gene from human bladder detrusor smooth muscle and construct its antisense eukaryotic expression vector. METHODS: The ¦Â3ª²AR fullª²length cDNA was cloned and amplified from human detrusor muscle cells through RTª²PCR (BamHI and ClaI sites were added into the 5¡ä end of upstream primer, and HindIII into downstream) and subcloned into clone vector (pUC18). The target gene was then cut from ClaI/HindIII sites of pUC18 with restriction endonuclease and inserted inversely into pLNCX vector. Finally, the constructed ¦Â3ª²AR gene antisense expression vector was identified though restriction endonuclease analysis and sequencing. RESULTS: Target gene of about 1.2 kb was successfully cloned into retroviral vector pLNCX. The sequence of cloned ¦Â3ª²AR fullª²length cDNA was identical to the reported one in GenBank. The constructed antisense eukaryotic expression vector was proved the same as designed by restriction endonuª²clease analysis and sequencing. CONCLUSION: ¦Â3ª²AR fullª²length cDNA was cloned from human bladder smooth muscle and their antisense eukaryotic expression vectors were constructed successfully. ......