Construction and Identification of RNA Interference Vector Targeting NCAM140 Gene
Author(s): CHEN Fang-fang, LI Li, CHEN Hui-zhen, XIAO Cheng-hua, GAO Dian-shuai, Department of Neurobiology, Xuzhou Medical College, Department of Pathophysiology, Xuzhou Medical College, Department of Neurology, Affiliated Hospital, Xuzhou Medical College
Pages: 251-255+285
Year: 2014 Issue: 2
Journal: Biomagnetism
Keyword: NCAM140; RNA interference; Plasmids;
Abstract: Objective: To construct RNA interference plasmid targeting NCAM140, it provided stable RNA interference plasmid for NCAM140 study involved in cell signaling pathway, function of molecular biology and NCAM140-targeting gene therapy. Methods: 4 gene sequences and 1 non-sence control gene sequence consistent with the screening features described in previous articles using open websites were designed, and the sequences were synthesized with the GenePharma Company. The sequences were amplified in Bacterium coli after gene recombination with pGPU6/GFP/Neo plasmid, and were named as pSi-nca1, pSi-nca2, pSi-nca3, pSi-nca4 and pSi-control respectively. Those plasmids were transfected into E. coli competent cells and positive clones were selected for DNA sequencing. After sequencing identification, the positive clones were transfected into MN9D cells. The GFP positive percentages(GFP positive cells versus total number of MN9D cells) were used to determine the transfer efficiency of these plasmids, and the inhibitory efficiency of these plasmids were confirmed by western blot. Results: shRNA was inserted into plasmid pGPU6/GFP/Neo correctly confirmed by digestion and DNA sequencing. The protein level of NCAM140 in MN9D cells at 24 hr after transfected into pSi-nca4 Plasmid is depressed(P <0.001). The most efficient plasmid was pSi-nca4. The pSi-nca4 transfection efficiency was 62 %. Conclusion: RNA interference plasmid targeting NCAM140 is successfully constructed, it provides stable RNA interference plasmid for the research of NCAM140 participating in cell signaling pathway, and offers favorable tool for the research of NCAM 140 biological effects and NCAM140-targeting gene therapy.
Pages: 251-255+285
Year: 2014 Issue: 2
Journal: Biomagnetism
Keyword: NCAM140; RNA interference; Plasmids;
Abstract: Objective: To construct RNA interference plasmid targeting NCAM140, it provided stable RNA interference plasmid for NCAM140 study involved in cell signaling pathway, function of molecular biology and NCAM140-targeting gene therapy. Methods: 4 gene sequences and 1 non-sence control gene sequence consistent with the screening features described in previous articles using open websites were designed, and the sequences were synthesized with the GenePharma Company. The sequences were amplified in Bacterium coli after gene recombination with pGPU6/GFP/Neo plasmid, and were named as pSi-nca1, pSi-nca2, pSi-nca3, pSi-nca4 and pSi-control respectively. Those plasmids were transfected into E. coli competent cells and positive clones were selected for DNA sequencing. After sequencing identification, the positive clones were transfected into MN9D cells. The GFP positive percentages(GFP positive cells versus total number of MN9D cells) were used to determine the transfer efficiency of these plasmids, and the inhibitory efficiency of these plasmids were confirmed by western blot. Results: shRNA was inserted into plasmid pGPU6/GFP/Neo correctly confirmed by digestion and DNA sequencing. The protein level of NCAM140 in MN9D cells at 24 hr after transfected into pSi-nca4 Plasmid is depressed(P <0.001). The most efficient plasmid was pSi-nca4. The pSi-nca4 transfection efficiency was 62 %. Conclusion: RNA interference plasmid targeting NCAM140 is successfully constructed, it provides stable RNA interference plasmid for the research of NCAM140 participating in cell signaling pathway, and offers favorable tool for the research of NCAM 140 biological effects and NCAM140-targeting gene therapy.
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