[Construction of pGL3-SM22-SCAP (D443N) eukaryotic expression vector and its expression in CHO cells].
The experiment was designed to investigate the function of SREBP cleavage-activating protein (SCAP) mutant (D443N) by constructing an eukaryotic expressive vector using a smooth muscle specific promoter SM22 (pGL3-SM22-SCAP(D443N)). SM22 promoter (pSM22) was amplified from genome DNA of mice by nested PCR, and then cloned into pMD-T vector. The SM22 promoter fragment released from the vector by Kpn I and Hind III digestion was sub-cloned into pGL3-control-Luc vector, to form pGL3-SM22-Luc. The activity of pSM22 in human vascular smooth muscle cells (VSMCs) was tested using Dual-Luciferase Reporter System. SCAP(D443) mutant amplified from plasmid pTK-HSV-SCAP(D443N) and pSM22 from mice liver were cloned into pGL3-control vector to construct pGL3-SM22-SCAP(D443N) which was transfected into Chinese hamster ovary cells (CHO) to test SCAP(D443) expression by real-time PCR and Western blot. The sequence and construction of pGL3-SM22-SCAP(D443N) were correct. SM22 promoter activity initiated the expression of luciferase in VSMCs and also drove SCAP(D443) expression in transfected CHO cells. The pGL3-SM22-SCAP(D443N) eukaryotic expression vector was successfully constructed and the recombinant vector provides a powerful approach in investigating the function and regulation of SCAP and also in producing vascular smooth muscle specific SCAP transgenic mice.
Wang
Yuanyuan
Y
Center for Lipid Research, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400016, China.
Hu
Jieli
J
Cui
Jing
J
Huang
Ailong
A
Ruan
Xiongzhong
X
Chen
Yaxi
Y
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000502:physiology; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D008840:Microfilament Proteins / Q000235:genetics*; D009124:Muscle Proteins / Q000235:genetics*; D050505:Mutant Proteins / Q000096:biosynthesis / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D050505:Mutant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C102196:SREBP cleavage-activating protein; C051615:transgelin