microRNA has an important regulatory role in cell proliferation and differentiation. The microRNA-378-1 precursor was amplified by PCR from Large White (Sus scrofa) genomic DNA and was digested by Xho I and then ligated into Xho I digested vector pEGP-miR to produce the recombinant vector pEGP-miR-378-1. This vector was transfected to porcine fetal fibroblasts cells (PEF, porcine embryo fibroblast) and its transfection efficiency and expression were detected by fluorescence observation and RT-PCR, respectively. The results showed that pEGV-miR-378-1 vector was constructed successfully with high transfection efficiency. Further RT-PCR results showed that the expression of microRNA-378-1 was increased significantly compared with control groups (P<0.01). This will provide technological platform for producing transgenic animals to further study the function of microRNA-378-1.%microRNA对细胞的增殖和分化有着重要的调节作用,从大白猪(Sus scrofa)基因组DNA上扩增microRNA-378-1前体序列,经Xho Ⅰ酶切后回收相应片段,连接到pEGP-miR载体中,构建重组载体pEGP-miR-378-1,转染猪胎儿成纤维细胞系(PEF),通过荧光观察转染效率及报告基因的表达效率,利用实时荧光定量PCR(QRT-PCR)测定细胞内microRNA-378-1的表达活性.结果表明,成功地构建了重组载体pEGP-miR-378-1;荧光观察转染后的细胞,显示有较高的转染效率,报告基因有较高的表达效率；QRT-PCR结果显示,实验组和对照组microRNA-378-1表达显著提高,实验组和对照组细胞间差异极显著(P＜0.01)；为制备转基因动物研究microRNA-378-1功能提供技术平台.
展开▼
