南方医科大学学报

南方医科大学学报 ›› 2014, Vol. 34 ›› Issue (09): 1359-.

• • 上一篇    下一篇

慢病毒干扰小鼠附睾特异的meClps基因可降低精子的运动能力

廉滋珍，曹佐武，陈冉，陈雷，薛樱子，秦俊文，齐绪峰，张春雪，禹艳红   

  • 出版日期:2014-09-20 发布日期:2014-09-20

Lentiviral vector-mediated RNA interference of mouse epididymis-specific meClps gene
lowers mouse sperm mobility

  • Online:2014-09-20 Published:2014-09-20

PDF

1146

摘要： 目的筛选能有效干扰小鼠附睾特异的类辅脂酶meClps基因表达的RNA靶点，并通过慢病毒介导的RNAi敲低meClps
表达后分析对精子运动的影响。方法构建真核表达载体pDsRed2.0-C1-meClps中并转染NIH-3T3细胞，用meClps抗血清检
测meClps蛋白的表达。针对meClps基因的序列设计并合成3对靶向干扰meClps表达的寡核苷酸序列，构建重组慢病毒载体
pRNAT-U6.2/lenti-RNAi-251、224和286。将重组慢病毒干扰质粒分别与pDsRed2.0-C1-meClps共转染到NIH-3T3细胞，半定
量PCR和Western blotting检测meClps基因在mRNA和蛋白水平的变化。将筛选得到的干扰效果最好的慢病毒载体包装慢病
毒，注射到小鼠附睾头部组织后分析对附睾尾部精子运动性能的影响。结果构建了体外表达meClps 蛋白的
pDsRed2.0-C1-meClps 表达载体和靶向meClps 基因的慢病毒RNAi 载体。靶向meClps 的RNA 干扰载体对转染
pDsRed2.0-C1-meClps后的NIH-3T3 细胞中的meClps 的mRNA和蛋白表达都有抑制，但以转染pRNAT-U6.2/lenti-RNAi-251
对meClps的抑制效果最明显。包装后的慢病毒注射到小鼠附睾头部后附睾尾部组织中的精子运动速率降低（P<0.05）。结论
筛选出RNAi-251能有效干扰meClps表达，通过慢病毒介导的RNAi敲低meClps表达后精子运动性能降低。

Abstract: Objective To analyze the effect of small interfering RNA (siRNA) targeting mouse epididymis-specific colipase-like
(meClps) gene on mouse sperm mobility. Methods The eukaryotic expression vector pDsRed2.0-C1-meClps was constructed
and transfected into NIH-3T3 cells, and the protein expression was detected with anti-meClps serum. Three interfering
sequences targeting meClps (RNAi-251, 224 and 286) were inserted into lentiviral vectors pRNAT-U6.2/lenti, which were
co-transfected with pDsRed2.0-C1-meClps into NIH-3T3 cells. The RNA interfering efficiency was confirmed by
semi-quantitative PCR and Western blotting. The lentivirus, packed with the lentiviral vector with the highest interfering
efficiency, was injected into the caput tissues of mouse epididymis, and its effect on sperm mobility of the cauda epididymis
was evaluated. Results All the 3 lentiviral RNAi vectors targeting meClps could inhibit the mRNA and protein expressions of
meClps, among which pRNAT-U6.2/lenti-RNAi-251 had the highest interfering efficiency. The lentivirus packed with
pRNAT-U6.2/lenti-RNAi-251 significantly reduced the path velocity of cauda sperm after injection into the caput epididymis
of the mice (P<0.05). Conclusion Knock-down meClps expression by lentiviral-mediated RNA interference can lower sperm
mobility of mice.