利用RNA干扰技术构建PYGO1基因RNA干扰(RNAi)的真核表达质粒(pSUPER -PYGO1)。首先, 针对 PYGO1 cDNA序列设计并化学合成一对编码短发夹RNA序列, 经退火插入到由BglⅡ 和XhoⅠ酶切的pSUPER 质粒上构建重组RNAi质粒pSUPER-PYGO1。通过XbaⅠ酶切鉴定及测序分析验证构建效果, 将正确构建的质粒转染大鼠心肌细胞系H9c2建立产生逆转录病毒的细胞克隆。通过RT-PCR和Western blot检测pSUPER-PYGO1对H9c2细胞中PYGO1 mRNA和PYGO1蛋白的干扰效果。pSUPER-PYGO1质粒经酶切鉴定及测序分析, 发现59nt寡核苷酸成功插入到预计位点, 且序列完全一致; RT-PCR和Western blot检测结果显示转染 pSUPER-PYGO1的细胞中PYGO1 mRNA和PYGO1蛋白量明显降低。因此, 靶向PYGO1的 pSUPER RNAi 载体构建成功, 为进一步从分子水平探讨PYGO1在心脏发育中的功能奠定了基础。

The eukaryotic expression vector of pSUPER-PYGO1 that can inhibit PYGO1 gene expression was constructed. Firstly, a pair of oligo nucleotides coding for short hairpin RNA targeting gene PYGO1 were chemically synthesized, then annealed and inserted into pSUPER plasmids digested with BglⅡ　and XhoⅠ to construct the recombinant RNAi plasmid pSUPER-PYGO1. Recombinant pSUPER-PYGO1 plasmid was identified by enzyme digestion and sequencing analysis. The H9c2 cells were transfected with the recombinant plasmid. The interferential effect was demonstrated by RT-PCR and Western blot. The result of enzyme digestion and sequencing analysis demonstrated that 59 nt oligonucleotides had been inserted successfully into the vector. RT-PCR and Western blot showed that pSUPER-PYGO1 can inhibit the transcription of PYGO1 mRNA and the expression of PYGO1 protein. The results of this studies implied that the pSUPER RNAi vector targeting human heart developmental candidate gene PYGO1 were successfully constructed and it established a foundation for the study on the molecular function of PYGO1.