Evaluation of promoter activity by using single cell time course analysis

Abstract

By using the time lapse of both phase-contrast and fluorescent images of single cells, we investigated the promoter activity. HeLa cells were transfected with EGFP, which encodes EGFP under the control of CMV promoter or cMyc binding site, in the presence of Lipofectamin™ LTX. After transfection with EGFP, phase-contrast and fluorescent images of cells in each well were recorded at intervals of 10min for 24 hours by using a Programmable Cellular Image Tracer. The strong correlation between cell division and the onset timing of gene expression was not influenced by the transfected gene. On the contrary in the case of pMycEGFP, the intercept of regression line was 54.685 min and that was 562.82 min in the case of pCMVEGFP. Intercept means the total requirement time from nuclear entry to folding of EGFP. It suggested that the activity of promoter of CMV was about 10 fold higher than that of cMyc promoter. This result suggested that the activity of promoters were comparable by using single cell time course analysis. This result also indicates that by using single cell time course analysis, we could evaluate the time of every step by arranging transfection reagent, promoter, and gene.