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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front Med Chin    2009, Vol. 3 Issue (2) : 130-135     DOI: 10.1007/s11684-009-0030-9
RESEARCH ARTICLE |
Gene silencing efficiency of shRNA expression vectors targeting Cx43 in vitro
Cuihong ZHENG1, Yunxia WU2, Guangying HUANG1(), Wei WANG3
1. Institute of Integrated Traditional and Western Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China; 2. School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China; 3. Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
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Abstract  

Our previous studies showed that there were close relationships between connexin 43 (Cx43) and acupoints and meridians. In order to further investigate the effect of Cx43 in acupuncture treatment, RNA interference technique was used to construct small hairpin RNA (shRNA) expression vectors targeting Cx43 and identify the efficiency of RNA interference in NIH/3T3 cell lines for further use in vivo. Aiming directly at the two targets of Cx43 mRNA sequence of the rat and mouse homology region, we synthesized two pairs of complementary oligonucleotide strands in vitro. Double strands were formed after annealing, and then inserted into Pgenesil-1 plasmid expression vector. After identification by enzyme cutting and sequencing, the recombinant plasmids named P-Cx43-shRNA (1), P-Cx43-shRNA (2) and P-con-shRNA were transfected into the NIH/3T3 cells. Immunofluorescence and Western blot assays were used to detect the protein level of Cx43 after being screened by G418.The results of enzyme cutting and sequencing showed that we successfully constructed two shRNA expression vectors targeting Cx43, and a control expression vector for rat and mouse. Also, the Cx43 protein level was decreased by 73.5% (P< 0.01) and 10.8%, accordingly. The Cx43 protein level was not influenced by the transfection of P-con-shRNA. The outcomes demonstrate that the plasmid P-Cx43-shRNA (1) can specifically silence better the expression of Cx43 in NIH/3T3 cells, which offers an experimental evidence for further in vivo investigation.

Keywords RNA interference      connexin 43      small hairpin RNA (shRNA)      acupuncture     
Corresponding Authors: HUANG Guangying,Email:gyhuang@tjh.tjmu.edu.cn   
Issue Date: 05 June 2009
URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-009-0030-9     OR     http://academic.hep.com.cn/fmd/EN/Y2009/V3/I2/130
Fig.1  Enzyme cutting of the recombinant plasmids. M: DL2000 marker; 1: P-Cx43-shRNA (1); 2: P-Cx43-shRNA (1)-I; 3: P-Cx43-shRNA (1)-I; 4: P-Cx43-shRNA (2); 5: P-Cx43-shRNA (2)-I; 6: P-Cx43-shRNA (2)-I; 7: P-con-shRNA; 8: P-con-shRNA-I; 9: P-con-shRNA 9-I.
Fig.2  Sequencing of the recombinant plasmids. (a) P-Cx43-shRNA (1) group; (b) P-Cx43-shRNA (2) group; (c) P-con-shRNA group.
Fig.3  The expression of EGFP in the NIH/3T3 cells ( ′ 200). Twenty-seven h after transfection of the recombined plasmids, (a) P-Cx43-shRNA (1) group; (b) P-Cx43-shRNA (2) group; (c) P-con-shRNA group.
Fig.4  Indirect immunofluorescence for the expression of Cx43 in the NIH/3T3 cells. (a) normal control group; (b) P-Cx43-shRNA (1) group; (c) P-Cx43-shRNA (2) group; (d) P-Cx43-shRNA (2) group. Green fluorescence was FITC for Cx43 immunocytochemistry staining, and red one for nuclear PI counterstaining. (a1) - (d1) were single FITC images, and (a)-(d) were merged images for FITC and PI (immunofluorescent staining ′ 400).
Fig.5  Immunoblotting assay for Cx43 protein level detection in NIH/3T3 cells (= 3). : <0.01 as compared with normal control group.
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