小鼠MSTN基因RNA干涉载体的构建及干涉效率的检测

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。

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

To select the RNA interference(RNAi)sequence of mouse myostatin gene,the coding sequence(CDS)of mouse myostatin gene was cloned by RT-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 dramatic decline in the expression level of mouse myostatin gene,and the RNAi efficiency of pRNAT-M1 vector was highest than any other interference sequences.It is concluded that the interference sequence obtained from this study shows an obvious interference effect on the expression of MSTN gene.