Enhancement of Exogenous Gene Expression by Artificial Transcription Factor in CHO Cells

Abstract:

Using the amino acids 1-147 of the yeast transcriptional activator GAL4 as the DNA_binding domain and four tandem repeats of the 12_aa peptide (DALDDFDLDMLG)of the herpesvirus as the activation domain,an artificial transcription factor,GVP4,was constructed via the linkage of the nuclear localization signal sequence of SV40.And then,GVP4 was cloned into expression vector pcDNA3.1/Hygro (+).Various amounts of targeting sites of artificial transcription factor were linked to the upstream of promoter CMV in exogenous gene expression vector pcDNA3.1(+) that separately harbored EGFP cDNA and t_PA cDNA.The CHO cells were then co_transfected with GVP4 expression vector and EGFP or t_PA expression vector.The effect of GVP4 on exogenous gene expression was evaluated by measuring the fluorescence intensity of EGFP in CHO cells and the concentration of t_PA in the supernatant.GVP4 showed positive effect on the enhancement of exogenous gene expression in CHO cells integrated with targeting sites of artificial transcription factor.And,CHO cells integrated with 10 targeting sites of GVP4 was more favorable to foreign gene expression,which resulted in 2～3_fold increase in both EGFP and t_PA expressions.These results indicated that artificial transcription factor is potent in the enhancement of exogenous gene expression in mammalian cells.