siRNA真核表达载体构建及对细胞MSTN基因抑制的研究

肌肉生长抑制素是肌肉生长的负调控因子。本研究为了得到有效抑制绵羊MSTN基因的siRNA分子,设计8对siRNA寡聚核苷酸引物,经退火与pSilencerTM4.1-CMV neo连接构建siRNA真核表达载体,转染C2C12细胞后,利用RT-PCR及real time PCR检测干扰效果。结果显示,8条siRNA分子中有3条对MSTN基因有显著的抑制作用,其中psi-mstn-717和psi-mstn-986分子对MSTN mRNA抑制效率分别达到61%和53%。本研究证实载体表达siRNA能有效抑制细胞MSTN mRNA的表达,下一步筛选的siRNA将用于体内实验。

Suppression of Ovis myostatin Gene Expression with Vector-based RNA Interference Technique

To construct the expression vector of siRNA targeting myostatin gene of ovis, and to observe the inhibitory effect of the vector on myostatin gene expression in C2C12 cells .Methods: 55 base-pair oligonucleotide for small interfering RNA expression targeting MSTN gene was designed and chemically synthesized. After annealing, double oligonucleotide were inserted into the down-stream of CMV promotor of pSilencer4.1 to recombine pSilencer-mstn-siRNA plasmids. Eight siRNA expression vectors were transiently transfected into mouse fibroblast cell line C2C12 with lipofectamine-mediated transfection. RT-PCR and real time PCR was used to detect the expression levels of MSTN gene. Results: The recombined psi-mstn-717 and psi-mstn-986 vector significantly suppressed the expression of MSTN mRNA by 61% and 53% in cells. Conclusion: The constructed siRNA expression vector of MSTN gene can block the expression of MSTN gene in cell.