小鼠MSTN基因RNA干涉载体的构建及干涉效率的检测

Construction of RNAi vectors of mouse myostatin gene and the detection of its RNAi efficiency

为了筛选小鼠肌肉生长抑制素(MSTN)基因的干涉序列,根据GenBank中小鼠的MSTN基因序列,采用RT-PCR方法克隆获得MSTN基因序列,构建其真核表达载体pcDNA 3.1(+)-MSTN;根据MSTN基因序列设计合成3种MSTN基因干涉序列(M1、M2、M3),并构建相应的RNA干涉载体pRNAT-M1、pRNAT-M2、pRNAT-M3。将构建的真核表达载体pcDNA 3.1(+)-MSTN单独或分别与3种干涉载体共转染293GP细胞,检测干涉载体对MSTN基因的干涉效率。结果表明:试验成功克隆得到与GenBank中的序列同源性为99.91%的小鼠MSTN基因序列,并构建了真核表达载体pcDNA 3.1(+)-MSTN;与转染pcDNA 3.1(+)-MSTN后的293GP细胞中MSTN基因的相对表达量(1.000)相比,转染pRNAT-M1、pRNAT-M2、pRNAT-M3后MSTN基因相对表达量明显下降,pRNAT-M1的干涉效率最高。说明研究成功获得对MSTN基因表达具有明显干涉作用的干涉序列。

To select the RNA interference （RNAi）sequence of mouse myostatin gene, the coding sequence （CDS）of mouse myostatin gene was cloned by lit -PCR on the basis of the CDS of the myostatin gene （BC105674）published in GenBank, and the eukaryotic expression vector pcDNA 3.1 （ ＋ ） - MSTN was constructed. The RNAi vectors （ pRNAT - M1, pRNAT - M2 and pRNAT - M3 ） were constructed according to the interference sequences（ M1 ,M2 and M3） composed by mouse myostatin gene. The 293GP cells were transfected alone with the eukaryotic expression vector pcDNA 3.1 （ ＋ ） - MSTN or co transfected with the RNAi vector and the eukaryotic expression vector（ pcDNA 3.1 （ ＋ ） - MSTN ）, then the efficiency of the RNAi was detected. The result showed that 99.91% homology was found between the cloning sequence and the CDS from GenBank database for mouse myostatin gene, and the eukaryotic expression vector （pcDNA 3.1 （ ＋ ） - MSTN）was constructed. Furthermore, after 293GP cells were transfected by the RNAi vectors（ pRNAT - M1, pRNAT - M2 and pRNAT - M3 ）, there was a was dra matic decline in the expression level of mouse myostatin gene, and the RNAi efficiency of pRNAT - M1 vector was highest than any other inter- ference sequences. It is concluded that the interference sequence obtained from this study shows an obvious interference effect on the expression of MSTN gene.