Construction And Identification Of Stable Transfected Cell Line For Silencing ILK Gene

Objective: To study the function of integrin-linked kinase (ILK) in the cell, construct ILK gene shRNA lentiviral vector, and detect silencing effect of ILK in tongue squamous cell carcinoma cell line Tca-8113. Methods: the target to three sequences of ILK gene, designed and synthesis three pairs of oligo DNA, anneal to double-stranded DNA, and connect the linearized vector pENTR/U6 to generate shILK-LV lentiviral vectors, select positive clones and sequence. Using shILK-LV vector and packaging plasmid packaging Mix cotransfection of 293T cells to produce lentivirus. The expressing of green fluorescent protein (GFP) in 293T cell was measured to virus titer to determine the appropriate MOI value. Infect human tongue squamous cell carcinoma cell line Tca-8113 after obtaining recombinant lentivirus. ILK expression in Tca-8113 cells by qPCR and Western blot analysis. Results:The sequencing confirmed successfully constructed three ILK-shRNA lentiviral vectors, Tca-8113 cells were transfected by these respectively and shRNA-ILK-370 group ILK 's were detected by the method of sybr decreased apparently. ??Ct value of 0.268. With shRNA-ILK-370 lentiviral vector packaging virus, detect viral titer, and when MOI= 30-60, the positive cells was highest. Ineting Tca-8113 cells were then screened by antibiotic strains, the interference efficiency was 90.5% using qPCR, the ILK expression was significantly inhibited by Western blot. Conclusions: successfully constructed shILK-LV lentiviral vector and established a stable Tca-8113-shILK cell model, providing a solid foundation for the study of the role of ILK in tongue squamous cell signal transduction pathway.