Reliable transient promoter assay using fluorescein-di-beta-D-galactopyranoside substrate.
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 promoter-lacZ) and contains the Escherichia coli lpp 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 beta-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 beta-galactosidase produced in viable cells was also detected by fluorescein-di-beta-D-galactopyranoside (FDG) treatment followed by image analysis using inverted fluorescent microscopy, which allowed the transfection efficiency to be calculated, and the beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Institute for Protein Research, Osaka University, Suita, Japan.
K
K
Ikenaka
I
I
Fujino
N
N
Morita
Y
Y
Iwasaki
M
M
Miura
T
T
Kagawa
K
K
Nakahira
K
K
Mikoshiba
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D005452:Fluoresceins / Q000378:metabolism*; D005697:Galactosides; D005821:Genetic Techniques; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D009185:Myelin Proteins / Q000096:biosynthesis / Q000235:genetics*; D018991:Myelin Proteolipid Protein; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D005452:Fluoresceins; D005697:Galactosides; D009185:Myelin Proteins; D018991:Myelin Proteolipid Protein; D011993:Recombinant Fusion Proteins; C038848:fluorescein-digalactoside; D001616:beta-Galactosidase