[Inhibiting GDF-8 expression by retrovirus-based RNAi stably].
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.
Liu
Chaowu
C
School of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
Yang
Zhuo
Z
Zhao
Bin
B
Liu
Changmei
C
D049109:Cell Proliferation; D002478:Cells, Cultured; D015536:Down-Regulation; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D055435:Myostatin / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics*; D034622:RNA Interference; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics* / Q000378:metabolism
C525911:MSTN protein, human; D055435:Myostatin; D012333:RNA, Messenger; D034741:RNA, Small Interfering