Construction of a lentivirus vector containing Pax6 and its transfection of human bone marrow mesenchymal stem cells
10.3969/j.issn.2095-4344.2016.36.003
- VernacularTitle:慢病毒载体Pax6-EGFP构建及转染人骨髓间充质干细胞
- Author:
Jingwen LIU
;
Jing REN
;
Sheng CHEN
;
Baijun LI
;
Shenwen LIU
;
Bo QIN
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2016;20(36):5338-5344
- CountryChina
- Language:Chinese
-
Abstract:
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 cel s (BMSCs), gaining the BMSCs stably over-expressing Pax6 is crucial, which is also the basis of stem cel 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 cel s. The human BMSCs were transfected with recombinant lentivirus Pax6-EGFP as wel as lentivirus vector pHIV-EGFP, which was considered negative control group. The cel ular 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 successful y 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.
