高效抑制小鼠精子zfx基因shRNA分子的筛选

The shRNA Screen of High Effective Inhibition zfx from Type X Sperm of Mice

zfx是小鼠X性染色体上的特异基因，可能与X精子的发生有关，沉默或下调该基因的正常表达可影响X精子的正常发育，使后代性别比列发生偏移。本实验为了筛选对小鼠zfx基因有效的shRNA分子，利用RNAi技术针对X精子zfx基因组mRNA的不同外显子选取10个干扰靶位，成功构建了10个shRNA质粒表达载体；通过对体外培养的生精细胞zfx基因的干扰试验，并用qRT—PCR方法检测生精细胞中zfxmRNA的表达丰度。结果显示：有4个干扰质粒组对zfx基因表达抑制明显，抑制率为33．47％～92．61％，其中pqz1、pqz3和pqz4等3个表达质粒抑制效果最好，抑制率分别为70．1％（P〈0．05）、85％（P〈0．05）和92．61％（P〈0．01），表明这3个质粒能明显抑制小鼠生精细胞中zfx基因的表达。

The zfx is a special gene located on X chromosome in mice. Previously,it has been reported that this gene was related to the production of X sperm. And silencing or knocking-down zfx will influence X sperm development and further cause the differential sex ratio of the offsprings. The present study was carried out to screen the effective shRNA of zfx gene in mice, using RNAi method. Firstly,we selected 10 different interference targets according to the different exons of zfx, and constructed 10 shRNA plasmid expression vectors, successfully. Then, the shRNA was transfected to spermatogenic cells in vitro, respectively. Real-time PCR was used to detect the mRNA expression of zfx gene. Results showed that 3 shRNA palsime inhibited the expression of zfx gene significantly,with the inhibition rates varying from 70.1 % to 92.61%. Among the four shRNA plasmid, pqzl,pqz3 and pqz4 inhibited most significantly,with the rates of 70.1 %（P〈0.05） ,85%（P〈0.01） and 92.61 %（P〈0.01）, respectively. It is,therefore,indicated that these 3 shRNA plasmid expression vectors can significantly inhibit the expression of zfx gene in spermatogenic cells of mice.