Abstract:

Objective  To investigate the characteristics of the erythroid differentiation-related factor (EDRF) promoter, different lengths of EDRF promoters were cloned to drive GFP (green fluorescence protein, GFP) expression. Methods  Different lengths of EDRF promoters（697, 496, 484, 372 and 283bp）were amplified by PCR. Then, the promoters were cloned into pcDNA-GFP vectors to drive GFP expression in vitro. After NIH3T3 and MEL cells were treated with the vectors, the activation of the promoter was analyzed by microscopy and FACS analysis. Results   The 372bp promoter(-116~+256bp) was found to be the most effective one to drive GFP expression in NIH3T3 and Mel cells under microscopy. Moreover, the GFP positive rate of the 372bp promoter group in Mel cells was obviously higher than that of other promoter groups by flow cytometry analysis(P＜0.01). The GFP positive rate of the 372bp promoter group in NIH3T3 cells was (31.0±0.7)%, higher than that of other promoter groups (P＜0.01). While GFP positive rates of other promoter groups in NIH3T3 cells were more than 20%, which demonstrated that gene expression driven by the EDRF promoter was not specific in NIH3T3 cells, but specific in Mel cells. Conclusions  The 372bp EDRF promoter (-116~+256bp) was the more effective one to drive GFP expression,  which could be further used to study gene therapy for anemia.

Key words: Erythroid differentiation-related factor; Promoter; Gene expression; Green fluorescence protein