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Published on: June 4, 2012
[Construction of sense and antisense eukaryotic expression vector of novel gene Collectrin and its function in cell
Hong Zhang1, Xiang-ling Wang, Yan-ling Zhang
1Department of Nephrology, Peking University First Hospital, Beijing 100034, China. hongzh@bjmu.edu.cn
Objective:
To construct sense and antisense eukaryotic expression vector of novel gene Collectrin and identify its function in cell growth.
Methods:
The open reading frame of Collectrin was amplified by PCR and inserted into pcDNA3.1/V5-His plasmid. The recombinant plasmid was identified by restriction enzyme analysis and sequencing analysis. The recombinant plasmid was transfected into M-1 cell by using lipofectin mediation after being identified by restriction enzyme analysis and sequencing analysis. RT-PCR and Western blot were performed to identify the expression of Collectrin. Beta-Gal staining was used to define the effect of transfection. The growth of M-1 cells was examined by MTT and cell counting.
Results:
Compared with control group, the expression of Collectrin was decreased significantly at both nucleotide and protein levels transfected by antisense vector, but elevated in sense group. The cell growth was blocked after being transfected by antisense plasmid.
Conclusion:
The sense and antisense eukaryotic expression vector of novel gene Collectrin was successfully constructed. Collectrin was one of basic factors in cell growth.
