Expression Of Exogenous BFGF In CHO-K1 Using Lentiviral Expression System Based On Gateway Technology

Objectives: To study and establish the stable CHO cell line expressing exogenous mouse-derived bFGF integrated into its genomic DNA by the lentiviral expression system based on gateway technology.Methods: Firstly, the attB-bFGF-attB gene was obtained by PCR, cDNAs from C2C12 cells as the templates, according to the gateway technology, secondly, the expression construct was constructed through BP and LR reactions by the gateway technology ; thirdly, the pseudoviral particles containing the expression construct was generated by lentiviral packaging system in 293FT cells; Forthly, the pseudoviral particles infected CHO-K1 cells; lastly, the expression and activity of bFGF were assayed by PCR, electrophoresis, Western blotting and the MTT method at the levels of genome, cDNA and protein.Results: ( 1 ) The entry and expression constructs was obtained by gateway technology; (2) The pseudoviral particles containing the expression construct was produced, and have infected the CHO-K1 cells successfully, and the monoclonal cells was screened; (3 ) the assays for bFGF at the levels of genome, cDNA and protein by PCR, electrophoresis and western blotting demonstrated exogenous bFGF has been integrated into the genomic DNA of the CHO-K1,and bFGF is expressed at the higher level in the CHO-K1 cells with exogenous bFGF gene, and its activity is perhaps correspondingly increased by the assay of the MTT method.Conclusions: (1) The lentiviral expression system based on the gateway technology has been successfully constructed in our lab; (2) the exogenous bFGF gene has been successfully integrated into the genomic DNA of the CHO-K1 cells, and expressed stably; (3) bFGF in the CHO-K1 with exogenous bFGF gene is expressed at the higher-level compared with that in the CHO-K1 without exogenous bFGF gene, and also suggesting the secretion of bFGF is perhaps increased.