Reliable Transient Promoter Assay Using Fluorescein-di-β-D-galactopyranoside Substrate

Abstract

The promoter region of the mouse myelin proteolipid protein (PLP) gene was cloned into a promoter testing vector, pIP111. The pIP111 vector is a promoterless derivative of pCH110 (SV40 early region promoterlacZ) and contains the Escherichia coli Ipp transcription terminator sequence at the 5′ end of the cloning site. The newly constructed PLP-lacZ fusion plasmid (pWP) was transfected into PLP-nonproducing NIH-3T3 fibroblasts or PLP-producing C6 cells. When the measured β-galactosidase activity in the pWP-transfected cells was normalized to the pCH110-transfected cells (an appropriate control if the SV40 early region promoter functions constitutively in various cell lines), the results suggested that the promoter region of the PLP gene contains the information necessary for initiation of transcription in a C6 cell-specific manner. However, the β-galactosidase produced in viable cells was also detected by fluorescein-di-β-D-galactopyranoside (FDG) treatment followed by image analysis using inverted fluorescent microscopy, which allowed the transfection efficiency to be calculated, and the β-galactosidase activity obtained by the regular ONPG method was normalized with the value obtained. This procedure indicated that the promoter region of the PLP gene did not show C6-specific expression, because the SV40 early-region promoter was 10 times more active in NIH-3T3 cells than in C6 cells. Thus, the standard experiment gave misleading results. As our detection method is simple and can be used to analyze the promoter activity in a single cell, many applications should be possible. As an example, we show that fluorescence is detected only in GFAP-producing C6 cells when pIP111 with the promoter region of GFAP (which had been shown to contain elements for its tissuespecific expression) was introduced into C6 cells or GFAP-nonproducing NIH-3T3 cells.