Regulation of the Escherichia coli lac operon expressed in human cells.
We have investigated the use of various Epstein-Barr virus (EBV)-based vectors bearing the two components of the Escherichia coli lac operator-repressor (lacO, lacI) complex. Our aim was to develop a model system of gene expression by looking at the transcription of the bacterial beta-galactosidase coding gene (lacZ) in 293 human embryonic kidney cells. Several vectors have been built carrying different promoters upstream of the lacI and lacZ genes and in which natural or synthetic operator sequences were inserted in the 5' part of the lacZ gene. In transient expression assays we achieved efficient lacZ gene repression which could be released by the specific inducer isopropyl beta-D-thiogalactoside (IPTG). A stable transformed cell line carrying two EBV-derived plasmids with the building blocks of the lac operator/repressor system was established. This cell line allowed us to achieve a wide range of lacZ gene regulation. In this cell line IPTG alone could remove the repression to trigger a 5-fold increase of lacZ expression. Heavy metal ions, which induced the mouse metallothionein I promoter located upstream of the lacZ gene, added together with IPTG gave rise to a 40-fold induction of lacZ expression.
Biard
D S
DS
Laboratory of Molecular Genetics, UPR 42, CNRS, Villejuif, France.
James
M R
MR
Cordier
A
A
Sarasin
A
A
D001483:Base Sequence; D002104:Cadmium / Q000494:pharmacology; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D005822:Genetic Vectors / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000235:genetics; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology*; D007763:Lac Operon / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D015032:Zinc / Q000494:pharmacology; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D012097:Repressor Proteins; D002104:Cadmium; D007544:Isopropyl Thiogalactoside; D015500:Chloramphenicol O-Acetyltransferase; D001616:beta-Galactosidase; D015032:Zinc