慢病毒载体Pax6-EGFP构建及转染人骨髓间充质干细胞

Construction of a lentivirus vector containing Pax6 and its transfection of human bone marrow mesenchymal stem cells

BACKGROUND:Pax6 gene plays an important role in eye development and differentiation, and to study how it regulates the differentiation of human bone marrow mesenchymal stem cells (BMSCs), gaining the BMSCs stably over-expressing Pax6 is crucial, which is also the basis of stem cell replacement therapy. OBJECTIVE:To construct a lentivirus vector containing Pax6 and detect the expression of Pax6 in transfected human BMSCs. METHODS:Pax6 gene was extracted using PCR. After its connection with lentivirus vector pHIV-EGFP, it was then packaged by 293T cells. The human BMSCs were transfected with recombinant lentivirus Pax6-EGFP as well as lentivirus vector pHIV-EGFP, which was considered negative control group. The cellular morphology was observed by a fluorescence microscope, and the mRNA expression of Pax6 was detected by real-time PCR. RESULTS AND CONCLUSION:The recombinant lentivirus Pax6-EGFP was constructed successfully with a titer of 3×109 pfu/L. After the transfection, both the green fluorescent protein and Pax6 gene were expressed detected using fluorescence microscope and real-time PCR, showing that the method of lentiviral transfection is a safe and effective way to modify BMSCs.