Inducible expression of bcl-2 by the lac operator/repressor system in MDCK cells.
The lac operator/repressor-inducible system was utilized to dissect the biological consequences of human bcl-2 gene expression in Madin-Darby canine kidney (MDCK) cells. Cells were made transgenic for a constitutively expressed lacI gene, encoding lac repressor, and the bcl-2 gene that had been inserted downstream of a simian virus 40 (SV40) promoter containing the lac operator sequence. The expression of the bcl-2 gene could therefore be repressed to basal level by binding of lac repressor to the lac operator sequence in proximity to this SV40 regulatory region and be specifically activated by administration of the lactose analog isopropyl-beta-D-thiogalactoside (IPTG). We showed that expression of bcl-2 gene could be induced by 0.01 mM IPTG, and the maximal induction was obtained at 1 mM. With the treatment of IPTG, the Bcl-2 protein could be induced within 6 h. Moreover, the IPTG-inducible expression of Bcl-2 protein is a reversible process. Finally, functional assays revealed that IPTG-induced expression of bcl-2 gene conferred partial or complete resistance to homeless cell death or confluent cell death, respectively. The inducible expression system should be particularly useful for dissecting the effect of bcl-2 in phenotypic or morphological changes of MDCK cells.
Lin
H H
HH
Department of Physiology, National Cheng Kung University Medical College, Tainan, Taiwan.
Yang
T P
TP
Jiang
S T
ST
Liu
H S
HS
Tang
M J
MJ
D000818:Animals; D017209:Apoptosis; D002460:Cell Line; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D007763:Lac Operon; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D012097:Repressor Proteins; D014162:Transfection
D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins; D007544:Isopropyl Thiogalactoside