Wnt-3a/eGFP双表达基因慢病毒载体的构建及其在大鼠骨髓间充质干细胞中的表达

目的构建双表达基因慢病毒载体pLV.EX3d.P/puro-EF1A>Wnt-3a>IRES/eGFP,用携带目的基因Wnt-3a及示踪基因增强型绿色荧光蛋白(eGFP)的慢病毒转导大鼠骨髓间充质干细胞(rat bone marrow mesenchymal stem cells,rBMSCs),建立能够持续激活Wnt信号通路的体外细胞模型。方法基于GatewayTM技术构建双表达基因慢病毒载体,经293FT包装并释放出病毒转导rBMSCs,检测Wnt-3a以及β-catenin的表达水平。结果经测序证实目的基因Wnt-3a及示踪基因eGFP片段按正确方向重组入目的载体中。用含Wnt-3a/eGFP的病毒上清转导rBMSCs,转导率超过85%。RT-PCR证实rBMSCs-Wnt3a过表达wnt-3a和β-catenin。结论携带目的基因Wnt-3a及示踪基因eGFP的慢病毒可以稳定转染rBMSCs,构建了能够持续激活Wnt信号通路的体外细胞模型。

Construction of Wnt-3a / eGFP Co-expression Recombinant Lentiviral Vector and Its Expression in Bone Marrow MesenchymalStem Cells of Rats

Objective To construct Wnt-3a and eGFP co-expression recombinant lentiviral vector pLV.EX3d.P/puro-EF1A〉 Wnt3-a〉IRES/eGFP; to use lentivirus carried with the target gene Wnt-3a and tracer gene eGFP to transduct rBMSCs; to establish the model to activate the Wnt signaling pathway continuously in vitro and observe its effect on rBMSCs. Methods Co-expression lentiviral vector was constructed based on Gateway technology, packaged with 293FT followed by transducting into rBMSCs, polymerase chain reaction （RT-PCR） to observe the mRNA expression level of Wnt-3a and fl-catenin. Results The target gene of Wnt-3a and tracking gene of eGFP was transferred into pLV.Des3d.P/puro vector correctly and confirmed by restriction analysis. Viral supematant containing Wnt-3a/eGFP were transduced into rBMSC with more than 85%. RT-PCR confirmed over-expression of Wnt-3a and β-catenin. Conclusion A stable rBMSCs isolated cultrued system was establlished, lentivirus carded with the gene Wnt-3a and tracer gene eGFP can be transducted into rBMSCs stably, to establish the model which can activate the Wnt signaling pathway continuously in vitro.