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。
Objective To analyze the effect of small interfering RNA （siRNA） targeting mouse epididymis-specific colipase-like （meC1ps） gene on mouse sperm mobility. Methods The eukaryotic expression vector pDsRed2.0-Cl-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-Cl-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/Ienti-RNAi-251 sigmficantly 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.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2014年第9期1359-1364,共6页
Journal of Southern Medical University
基金
广州市珠江科技新星专项(2013J2200026)
关键词
RNA干扰
附睾特异表达蛋白
meClps
慢病毒
精子运动
RNA interference
epididymis-specific protein
mouse epididymis-specific colipase-like gene
lentivirus
sperm mobility
