The lac operator/repressor inducible system was utilized to dissect the biological consequences of human bcl-2 gene expression in 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 SV 40 regulatory region and be specifically activated by administration of the lactose analogue isopropyl β-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 reveals that IPTG-induced expression of bcl-2 gene conferred partial or complete resistance to anoikis (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.
|出版狀態||Published - 1997 十二月 1|
All Science Journal Classification (ASJC) codes
- Molecular Biology