Smad3基因RNAi慢病毒载体的构建与鉴定

Construction and identification of lentiviral vector encoding shRNA against Smad3

目的构建Smad3基因RNAi慢病毒载体。方法针对已经筛选确定的Smad3基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经AgeⅠ和EcoRⅠ双酶切后的pGCSIL-GFP载体连接产生GC-shSmad3慢病毒载体,PCR筛选阳性克隆,测序鉴定。用GC-shSmad3、pHelper 1.0载体和pHelper 2.0载体共转染包装细胞293T细胞,包装产生慢病毒,以293T细胞GFP蛋白的表达水平测定病毒滴度。结果PCR和测序证实,构建出了Smad3 shRNA的慢病毒载体GC-shSmad3。包装慢病毒,浓缩病毒悬液的滴度为3×108TU/ml。结论成功构建Smad3基因RNAi慢病毒载体。

Objective To construct a lentiviral vector expressing small-hairpin RNA （shRNA） targeting Smad3 gene. Methods The targeting sequence of Smad3 gene which can be effectively silenced in RNA inference was confirmed in our previous study. The cDNA containing both sense and antisense Oligo DNA fragments of the targeting sequence was designed, synthesized and cloned into the pGCSIL-GFP vector. The obtained lentiviral vector containing Smad3 shRNA was confirmed by PCR and sequencing. 293T cells were cotransfected with lentiviral vector GC-shSmad3, pHelper 1.0 and pHelper 2.0. The titer of virus was tested according to the expression level of GFP. Results PCR and DNA sequencing demonstrated that the constructed lentivirus vec- tor GC-shSmad3 produced Smad3 shRNA. The titer of concentrated virus was 3 ×10^8TU/ml. Conclusion The lentivirus RNAi vector targeting Smad3 is constructed successfully.