长链非编码RNA AK005183干扰载体构建及慢病毒包装  

Construction of the Plamid Carrying Short Hairpin RNA Targeting Long Noncoding RNA AK005183

作　　者：张晓明[1] 安俊辉[1] 万一 胡源[1] 覃金洲 曾文先[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《家畜生态学报》2017年第10期15-19,共5页Journal of Domestic Animal Ecology

基　　金：国家自然科学基金项目(31072029;31272439)

摘　　要：为研究长链非编码RNA AK005183在精子发生中的功能,设计、合成了干扰AK005183表达的shRNA序列,并构建其干扰表达重组慢病毒载体CD513B-U6-AK005183,将干扰表达质粒及对照质粒分别转染NIH3T3细胞系,实时定量PCR检测CD513B-U6-AK005183质粒对AK005183的表达具有较好的干扰效果。利用第三代慢病毒包装系统,将CD513B-U6-AK005183、pGag-Pol、pRsv-rev、pVSV-G质粒共转染293T细胞系,包装得到滴度为5×106 TU/mL的干扰表达重组慢病毒。用CD513B-U6-AK005183慢病毒转导GC-1spg细胞系,检测到AK005183的表达下调64%。表明本研究成功构建并包装得到高效干扰长链非编码RNA AK005183表达的重组慢病毒,将为深入研究长链非编码RNA在精子发生过程中的功能奠定了基础。To study the function of long noncoding RNA AK005183 in sotide interfering AK005183 expression was designed and synthesized, and its interference combinant lentiviral vector CD513B-U6-AK005183 was constructed as well . Transfected NIH3T3 cells with interference expression plasmid to detect the interference efficiency of this shRNof AK005183 by real tme and quantitive PCR. By using the third generation lentivirus package system, the plasmids of CD513B-U6-AK005183, pGag-Pol, pRsv-rev, and pVSV-G was transfected to 293T cell llnes,an interference expression of recombinant lentivirus with titer of 5 × 10^6 TU/mL was aquired from the package. It was detected that the expression of AK005183 declined by 64% due to the transfection of GC-1spa cells lineswith lentivirus. This study successfully contructed and packaged the recombinant lentvirus which can highly and efliciently interfere with long noncoding RNA AK005183, providing a foudation for further research on function of long noncoding RNA in sperms.

关 键 词：长链非编码RNA AK005183 精原干细胞 RNA干扰 慢病毒 

分 类 号：S811.6[农业科学—畜牧学]