小鼠pLVX-Wnt3a-IRES-ZsGreen1慢病毒载体的构建及神经干细胞转染

Construction of mouse pLVX-Wnt3a-IRES-ZsGreen1 lentivirus vectors and transfection of neural stem cells

目的构建共表达小鼠Wnt3a(mWnt3a)与绿色荧光蛋白(GFP)慢病毒载体,感染神经干细胞(NSCs),观察mWnt3a在NSCs中的表达。方法利用同源重组技术将mWnt3a基因插入慢病毒载体pLVX-IRES-ZsGreen1,构建pLVX-Wnt3a-IRES-Zs-Green1慢病毒重组质粒,通过瞬时转染法包装出病毒上清,感染NSCs,设为Wnt3a-NSCs组;同时设GFP感染NSCs组(GFP-NSCs组)和未感染NSCs组(NSCs组)作为对照。免疫荧光染色法对Wnt3a-NSCs组NSCs进行nestin鉴定;Real-Time PCR检测各组细胞mWnt3a mRNA的表达;Western blotting检测各组细胞mWnt3a、β-catenin蛋白的表达。结果经限制性内切酶检测、基因测序和绿色荧光观察证实成功构建了携带mWnt3a基因的重组慢病毒,且慢病毒滴度达3×108TU/mL。Wnt3a-NSCs组NSCs在荧光显微镜下证实有绿色荧光,且nestin表达阳性。Real-Time PCR和Western blotting结果显示感染后7 d,Wnt3a-NSCs组mWnt3a mRNA和蛋白以及β-catenin蛋白均明显高于GFP-NSCs组和NSCs组(P<0.01)。结论成功构建了表达mWnt3a基因的慢病毒载体,在体外培养条件下可以成功转染NSCs。

Objective To construct lentivirus vectors with co-expression of mouse Wnt3a（mWnt3a） gene and green fluorescent protein（GFP）,transfect into neural stem cells（NSCs）,and observe the expression of mWnt3a in NSCs.Methods Gene recombinant technology was employed to clone mWnt3a gene to lentivirus vector pLVX-IRES-ZsGreen1 to construct a lentiviral vector pLVX-Wnt3a-IRES-ZsGreen1.Lentiviral vectors were packaged and the titer was determined.NSCs were transfected with the constructed pLVX-Wnt3a-IRES-ZsGreen1（Wnt3a-NSCs group） or pLVX-IRES-ZsGreen1（GFP-NSCs group）,with normal NSCs（NSCs group） as controls.NSCs of Wnt3a-NSCs group were identified by immunoflourence staining for nestin,the expression of mWnt3a mRNA in NSCs of each group was detected by Real-Time PCR,and Western blotting was applied to detect the expression of mWnt3a and β-catenin protein in NSCs of each group.Results It was confirmed by restriction enzyme digestion,DNA sequencing analysis and GFP expression detection that recombinant pLVX-Wnt3a-IRES-ZsGreen1 vector was successfully constructed,with virus titer up to 3×108 TU/mL.Not only green fluorescence but also expression of nestin were observed in NSCs of Wnt3a-NSCs group.Real-Time PCR and Western blotting revealed that the expression of mWnt3a mRNA and protein and β-catenin protein in Wnt3a-NSCs group was significantly higher than that in GFP-NSCs group and NSCs group 7 d after transfection（P〈0.01）. Conclusion The recombinant lentiviral vector carrying mWnt3a can be successfully constructed,and mWnt3a gene can be successfully transfected into NSCs in vitro.