Inhibiting GDF-8 expression by retrovirus-based RNAi stably.
- Author:
Chaowu LIU
1
;
Zhuo YANG
;
Bin ZHAO
;
Changmei LIU
Author Information
1. School of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
- Publication Type:Journal Article
- MeSH:
Cell Proliferation;
Cells, Cultured;
Down-Regulation;
Gene Expression Regulation;
genetics;
Humans;
Myoblasts;
cytology;
metabolism;
Myostatin;
antagonists & inhibitors;
biosynthesis;
genetics;
RNA Interference;
RNA, Messenger;
analysis;
metabolism;
RNA, Small Interfering;
genetics;
Retroviridae;
genetics;
metabolism
- From:
Chinese Journal of Biotechnology
2008;24(2):250-255
- CountryChina
- Language:Chinese
-
Abstract:
We cloned human U6 promoter from pAVU6 + 27 vector into pXSN to transcripe small RNA. Meanwhile, a shRNA targeting GDF-8 was cloned down-stream of the hU6 promoter to construct recombinant vector. Then the packing cell GP-293 was co-transfected the recombinant with pVSV-G to gernarate virus particle. Resistant C2C12 cell pools were screened using G418. Levels of mRNA and protein of GDF-8 were tested by Real-Time PCR and western blotting. Cell proliferation and cell cycle were analyzed using MTT and FACS. The expression of GDF-8 was dramatically decreased by the retrovirus-based system in C2C12 cells. Cells proliferated effectively after integrating the recombinant. The cells in G0/G1 phase decreased by 13.7%, while cells in S phase increased by 14.9%. In conclusion, the retrovirus-based RNAi could be used to stably silence GDF-8. It can be a powerful tool in curing muscle atrophy.
