An Improved Transfection Assay for Evaluating the Effects of Heavy Metals

Abstract

The transfection assay is an important tool for evaluating the health effects of industrial chemicals, with the reporter gene expression as an indicator. However, heavy metals often influence the expression of the reference plasmids used to correct variations in transfection efficiency between assay plates, reducing the reliability of this assay. We found that the target of heavy metals is the reporter, rather than the promoter used in the reference plasmid. Of the reporters we tested, luciferase (Luc) enzyme activity was affected by heavy metals, whereas gene product levels of the chloramphenicol acetyltransferase (CAT) or β-galactosidase (βGal) gene were not. Neither heavy metals nor extracts from cells exposed to heavy metals showed an effect when directly added to the Luc enzyme, suggesting that heavy metals act through an indirect mechanism. These data indicate that the use of CAT or βGal as a reporter is appropriate for a reliable assay of heavy metal effects.

Analysis of Cysteine and Histidine Residues Required for Zinc Response of the Transcription Factor Human MTF-1

“…CAT levels in cell lysates were analyzed using CAT-ELISA (Roche). In experiments using the reference plasmid pRSVL, luciferase activity was measured as described previously, 22,24) and relative reporter activity, i.e., CAT level normalized relative to luciferase activity, is indicated unless otherwise stated.…”

Evaluation of Transfected HEP-2 Cell Line Using ß-Galactosidase Reporter Assay System

“…In addition the widely usage of the gene for studying the activity of promoters which is depend on use of cell line for expression [8]. Other studies used this gene to evaluate the transfection methods or reagents in gene transfer [9].…”

Evaluation of Transfected HEP-2 Cell Line Using ß-Galactosidase Reporter Assay System

“…In addition the widely usage of the gene for studying the activity of promoters which is depend on use of cell line for expression [8]. Other studies used this gene to evaluate the transfection methods or reagents in gene transfer [9].…”