A hybrid promoter-containing vector for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells.
An efficient vector, designated as pCAGX, was designed for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells. It relied on the well-known TA-cloning principle, and utilized the CMV enhancer/chicken beta-actin/rabbit beta-globin (CAG) hybrid promoter instead of the classical CMV promoter to drive more efficient transgene expression in wider host cells. The specially designed cassette under CAG hybrid promoter contained two tandemly arrayed XcmI sites which were spaced by an additional EcoRV site. For direct cloning and expressing PCR-amplified ORFs, the T-vector was prepared by further digesting the EcoRV-linearized pCAGX with XcmI to produce T tails on both 3'-ends, which could efficiently minimize the non-recombinant background of T-vector and eliminate the necessity of selective marker genes such as LacZ that allowed blue/white screening. Various PCR fragments in length were prepared to verify the cloning efficiency by ligation with this vector, and GFP gene expression under control of the CAG hybrid promoter in different host cells was assayed by flow cytometry. The results indicated that this vector was higher efficient, especially suitable for cloning and expressing a number of interesting ORFs in parallel, and higher-level transgene expression in different mammalian cells was obtained than the reported vectors using the CMV promoter.
You
Lei-ming
LM
Key Laboratory of Animal Immunology of the Ministry of Agriculture, Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, 450002 Zhengzhou, People's Republic of China.
Luo
Jun
J
Wang
Ai-ping
AP
Zhang
Gai-ping
GP
Weng
Hai-bo
HB
Guo
Ya-nan
YN
Liu
Yun-chao
YC
Li
Qiao-mu
QM
Teng
Man
M
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007438:Introns / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D017452:Receptors, IgG / Q000235:genetics; D014162:Transfection
D017452:Receptors, IgG; D049452:Green Fluorescent Proteins