The amplification and cloning of promoters are regularly employed procedures to study the mechanism of gene regulation. In the present study we developed a method to construct T-vectors used for the direct and unidirectional cloning and analysis of promoters. These so-called T-vectors pEGFP-T and pGL3-T were derived from their parent promoterless vectors pEGFP-1 and pGL3-basic, respectively. To construct the T-vectors, an AhdI recognition site within the Ampr gene in pGL3-basic was silent mutated using overlap extension PCR. Then, a specially designed AhdI cassette was cloned into the respective parent vectors. The procedures of the T-vector construction involved a strategy to minimize the background of nonrecombinant transformants and to eliminate reverse orientation of the PCR products into the T-vectors. The cloning efficiencies of the two T-vectors were both above 85% when tested with a PCR product amplified from a sequence that was pre-confirmed to be able to initiate transcription, and moreover, the constructs harbored the inserts in a desired orientation at a >90% rate. In transient transfection assays, we demonstrated these T-vectors are functional. Thus, the present study provides an easy method to construct a series of T-vectors used for promoter characterization.

+More

gene regulation promoter characterization tvector construction ahdi recognition transfection nonrecombinant transformants amplification overlap extension tvectors direct and unidirectional cloning and analysis of promoters

B L, Wang X X, Li F, Zheng R, Liu J X, Quan H W, Jia H, Liang T, Deng S Y, Guo G, Guo J Y, Zhang M C, Qiu,.[Construction of T-vectors for the direct, unidirectional cloning and analysis of PCR-amplified promoters].. 41 (4),719-24.

B L, Wang X X, Li F, Zheng R, Liu J X, Quan H W, Jia H, Liang T, Deng S Y, Guo G, Guo J Y, Zhang M C, Qiu,"[Construction of T-vectors for the direct, unidirectional cloning and analysis of PCR-amplified promoters]." 41

B L, Wang X X, Li F, Zheng R, Liu J X, Quan H W, Jia H, Liang T, Deng S Y, Guo G, Guo J Y, Zhang M C, Qiu,"[Construction of T-vectors for the direct, unidirectional cloning and analysis of PCR-amplified promoters]."