人β-catenin-EGFP融合表达慢病毒载体构建及在毛囊干细胞中的表达

Objective To construct the lentivirus carrying human β-catenin-EGFP (enhanced green fluorescent protein) and observe its expression in human follicle stem cells. Methods The β-catenin gene sequence was amplified by RT-PCR from extraction of total RNA of human vascular endothelial cells. TA cloning technique was utilized to acquire gene subcloned pUCm-T-β-catenin. After transformation reaction, candidate clone was further analyzed by PCR and gene sequencing. Then the plasmid was transfected into FT293 cells. After identification by Western blotting, the plasmid was transfected into FT293 cells again for packaging. Infection titer was monitored by green EGFP expression. The expression of β-catenin-lentivirus in human follicle stem cells were observed under inverted fluorescence microscope. Results The β-catenin gene was cloned into the lentivirus successfully. The high expression of green fluorescence protein in FT293 cell line was found under fluorescent microscope. Viral titer checked by real-time PCR was about 2.0×10^8 TU/mL. When the multiplicity of infection (MOI) was 10, the infection efficiency of β-catenin-lentivirus in human follicle stem cells was nearly 80% after infection 48 h around. After 3 weeks of continuous observation, we found the infection efficiency still keeping in the range of 80%~90%. Conclusion The lentivirus expression vector for β-catenin was successfully constructed. It can steadily infect human follicle stem cells and the infection efficiency is considerable high.