关键词: 慢病毒表达载体, Islet-1 , 干细胞特异性分化, 心肌样细胞

Abstract: Object To construct lentiviral expression vectors targeting mouse gene Islet-1 and to study the effect of Islet-1 on stem cell differentiation. Methods Islet-1 gene was obtained by PCR and insected into pLenO-WPI Vector. The positive plasmid was selected and infected 293T cells with helper plasmid to produce recombinact lentivirus. Islet-1 expression and its related gene, gene and protein markers of heart, liver, bone, and nerves systems were measured by Real-time quantitative PCR and Western blot after C3H10T1/2 was infected by the lentiviruses. Results PCR and sequencing showed that the right DNA fragment and Islet-1 had been inserted and can be detected in both in gene and protein levels. Cardiac development related genes GATA-4, MEF2C; NKx2.5 increased in 1week and reached the highest level in 2 weeks, and cTnT reached a high level in 3 weeks and increasedover time. There were no expressions of gene and protein markers of liver, bone, brain. Conclusion Lentiviral expression vector carrying mouse Islet-1 gene was constructed. Islet-1 promoted C3H10T1/2 cells differentiate into cardiomyocyte-like cells specifically, that laid the foundation for further investigation of Islet-1.

Key words: Lentiviral expression vector, Islet-1, MSCS differentiation, Cardiomyocyte-like cells