Cloning of the PTEN/MMAC1 cDNA and construction of its expression plasmid
Jian Chen · Letters in Biotechnology · 2001
In order to obtain PTEN/MMAC1 cDNA and construct the bait plasmid of yeast two?hybrid system and its retroviral plasmid. About 1.2 kb DNA fragment was amplified from the 293 cell by RT?PCR. After automatic sequenced, the fragment was ligased with the vector pLexA to construct pLexA?PTEN/MMAC1. The recombinant plasmid was transformed into yeast EGY48(p8op?LacZ), and the expression of pLexA?PTEN/MMAC1 in the yeast was observed. Meanwhile, PTEN/MMAC1 cDNA fragment was ligased with retroviral vector pLXSN to construct pLXSNPTEN/MMAC1. Results is that the cDNA fragment by RT?PCR was same with reported PTEN/MMAC1 cDNA, and about 1 mm white yeast clone transformed with pLexA?PTEN/MMAC1 was grown in the selective medium after 3 days. About 1.2 kb DNA fragment was shown from the digested pLXSN?PTEN/MMAC1. Results indicated that the PTEN/MMAC1 cDNA has been cloned. pLexA?PTEN/MMAC1 can be used as a bait plasmid of yeast two?hybrid system. While the plasmid pLXSN?PTEN/MMAC1 make the base for further studying the tumor suppression function of PTEN/MMAC1.