PKCγ基因RNAi慢病毒载体的构建

目的 构建蛋白激酶Cγ(PKCγ)基因RNA干扰(RNAi)慢病毒载体.方法 针对已经筛选确定的PKCT基因RNAi有效靶序列,合成靶序列的短发夹RNA(shRNA)寡核苷酸序列(Oligo)DNA,退火形成双链DNA,与经Age Ⅰ和EcoR Ⅰ酶切后的pGCSIL-GFP载体[含U_6启动子和绿色荧光蛋白(GFP)]连接,转化DH5a大肠杆菌,挑选重组阳性克隆行PCR鉴定和DNA测序.用pGCSIL-GFP、pHelper 1.0和pHelper 2.0三质粒共转染293T细胞,包装产生慢病毒,逐孔稀释滴度法测定病毒滴度.结果 PCR鉴定结果显示,以经双酶切后未插入片断的pGCSIL-GFP空载体(PCR产物为306 bp)为对照,重组细菌克隆的PCR产物为343 bp(插入片段为37 bp),鉴定结果与预期相符.测序结果显示,合成的PKCT基因shRNA寡核苷酸链序列插入正确.包装慢病毒,浓缩慢病毒悬液的滴度为1×10~9 TU/ml.结论 成功构建了PKCT基因shRNA慢病毒载体.

Construction of lentiviral vector of RNA interference of PKCY gene

Objective To construct a lentiviral vector of RNA interference (RNAi) of PKCγ gene. Methods The effective sequence of siRNA targeting PKCγ gene was confirmed in our previous study. The complementary DNA containing both sense and antisense oligo DNA of the targeting sequence was designed, synthesized and cloned into the pGCSIL-GFP vector, which contained U_6 promoter and green fluorescent protein (GFP) . The resulting lentiviral vector containing PKCγshRNA was named lentivinis RNAi vector of PKCγ, and it was confirmed by realtime PCR and sequencing. 293T cells were cotransfected with lentiviral vector pGCSIL-CTP, pHelper 1.0 and pHelper 2.0. All virus stocks were produced by calcium phosphate-mediated transfection. The titer of virus was tested according to the expression level of GFP. Results PCR and DNA sequencing demonstrated that the lentivirus RNAi vector of PKCγ producing PKCγshRNA was constructed successfully. The titer of concentrated virus was 1 ×10~9 TU/ml. Conclusion The lentivinis RNAi vector of PKCy was constructed successfully.