[Construction of T-vectors for the direct, unidirectional cloning and analysis of PCR-amplified promoters].
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.
Wang
B L
BL
Li
X X
XX
Zheng
F
F
Liu
R
R
Quan
J X
JX
Jia
H W
HW
Liang
H
H
Deng
T
T
Guo
S Y
SY
Guo
G
G
Zhang
J Y
JY
Qiu
M C
MC
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000737:chemistry; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics*; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D014162:Transfection
D004262:DNA Restriction Enzymes