逆转录病毒载体介导的RNA干扰稳定抑制肌肉生长抑制素GDF-8的表达

Inhibiting GDF-8 Expression by Retrovirus-Based RNAi Stably

摘要:

为将肌肉生长抑制素的干扰序列表达盒hU6-siGDF-8有效导入肌成纤维细胞C2C12中, 以pAV-hU6 + 27载体为基础构建逆转录病毒载体pXSN-hU6-siGDF-8, 并使之与pVSV-G质粒共转染GP-293细胞, 用包装出的病毒粒子感染宿主细胞C2C12, G418筛选稳定整合逆转录病毒的抗性细胞库。2周后, Western Blotting和Real-Time PCR分析结果显示, 细胞内源性的GDF-8基因的表达得到了有效的抑制; MTT法和细胞流式仪分析表明, G418抗性细胞得到了更有效的增殖, 并且G0/G1期细胞数量减少了13.7%, S期细胞数量增加了14.9%。因此, 逆转录病毒载体的RNA干扰系统可以稳定抑制 GDF-8基因表达, 它将成为治疗肌肉萎缩疾病的一个强有力的工具。

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.