Abstract:

Objective To construct a lentiviral vector targeting the transgelin-2 （TAGLN2） gene in the mouse brain microvascular endothelial cell line bEnd.3 cells， and to identify its silencing effect. Methods Short hairpin RNA （shRNA） interference target sites were designed based on the coding region of the TAGLN2 gene. The amplified products were ligated into the GV493 lentiviral vector linearized by double digestion with EcoR Ⅰ and Age Ⅰ to construct the GV493-TAGLN2-shRNA recombinant lentiviral vector. The positive clones were screened by PCR and identified by sequencing. The GV493 control lentiviral vector and the GV493-TAGLN2-shRNA recombinant lentiviral vector were respectively transfected into the HEK293T cells， and the supernatants were collected and purified， and the viral titers were determined. The bEnd.3 cells were infected with a multiplicity of infection （MOI） of 100. After 48 h of infection， the complete medium containing 10 mg·L^-1 puromycin was replaced for screening and culture for 14 d. An inverted fluorescence microscope was used to observe the expression of green fluorescence protein （GFP） in the cells in two groups； real-time fluorescence quantitative PCR （RT-qPCR） method was used to detect the expression levels of TAGLN2 mRNA in cells in two groups； Western blotting method was used to detect the expression levels of TAGLN2 protein in the cells in two groups. Results The fluorescence microscope observation results showed that there was a strong GFP fluorescence signal in the transfected HEK293T cells， indicating that the lentiviral packaging system was successfully constructed. The titer of the GV493 control lentivirus was 5×1011 TU·L^-1， and the titer of the TAGLN2 interference lentivirus was 6×1011 TU·L^-1. After transfection， the bEnd.3 cells in two groups showed obvious GFP fluorescence signals， indicating that the stable transfected cell lines were successfully constructed. The RT-qPCR results showed that compared with GV493 control group， the expression level of TAGLN2 mRNA in the cells in GV493-TAGLN2-shRNA group was significantly decreased （P<0.01）. The Western blotting results showed that compared with GV493 control group， the expression level of TAGLN2 protein in the cells in GV493-TAGLN2-shRNA group was significantly decreased （P<0.01）. Conclusion A lentiviral vector targeting the TAGLN2 gene in bEnd.3 cells is successfully constructed， which can efficiently silence TAGLN2 expression.

Key words: Transgelin-2, Stably transfected cell line, Lentivirus, bEnd.3 cell, Short hairpin RNA