在活体小鼠中筛选IZP3基因RNAi有效靶位点的研究

ZP3作为精子结合的靶对象在卵母细胞受精中起着关键作用，也因此而成为研究哺乳动物受精机理的焦点。本研究参照新疆草原兔尾鼠卵透明带3（zonapellucida3 gene of Lagurus lagurus，IZP3）的mRNA，选择了针对IZP3 mRNA的3个区域合成寡聚核苷酸连接到干扰载体pGenesil-1上，构建了3个针对IZP3 mRNA的重组干扰载体，和pCDNA3-IZP3表达载体采用脂质体法共转染Hela细胞以及通过尾静脉大容量快速注射法（Hydrodynamics—based transfection method，HD法）共注射小鼠，半定量RT—PCR和real—timePCR检测Hela细胞和小鼠肝脏中IZP3mRNA的表达情况，以筛选干扰IZP3mRNA的有效靶位点。结果表明，通过HD法共注射干扰载体和表达载体后，外源基因mRNA在小鼠肝脏中的表达和共转染Hela细胞后在Hel。细胞中的表达情况是一致的，有2个干扰载体可以有效干扰IZP3mRNA的表达。研究还说明，利用HD法以小鼠作为实验材料筛选RNA干扰的有效靶位点是一条切实可行的方法。

Selecting the RNAi Efficiency Target Sites of IZP3 Gene by Mouse in vivo

As the combining target with sperms,ZP3 undergoes an important role in the fertilization of oocytes and therefore it has been the focus in studying the mechanism of mammalian. According to the sequence of the zona pellucida 3 gene of Lagurus lagurus (lZP3),three RNA interference recombinant vectors were constructed with pGenesil-1 aiming at lZP3 mRNA by synthesizing oligonucleotides. And then co-transfected into the Hela cells by Lipofectamine2000 and co-injected into the mice by hydrodynamics-based transfection method with the expression vector pCDNA3-lZP3. In order to select the efficient target sites of lZP3 for RNAi,the mRNA expression level of lZP3 gene in Hela cells and the mouse liver was detected by semi-quantative RT-PCR and real-time PCR. Results show that there are 2 interference vectors can interfere of the expression of lZP3 mRNA,and the mRNAs of the exogenous genes expressed in the mouse liver are coincident with those of in Hela cells after co-transfected with the interference vectors and expression vector. It also suggests that the mice can be the experimental materials for selecting the efficiency target sites of the RNA interference.