IPTG-Inducible Episomal Expression System for Exogenous Genes in Primate Cells

Abstract

An isopropyl beta-D-thiogalactopyranoside (IPTG)-inducible episomal expression system has been established for the human breast carcinoma cell line MCF7. This two-component system includes: (i) a primate cell-specific episomal vector, pEpiLac, that contains an IPTG-inducible promoter and (ii) a cell line derived from MCF7, MCF7/LAP5, which expresses the IPTG-dependent transactivator LAP267. Treatment of MCF7/LAP5 cells with IPTG results in efficient inducible expression of exogenous genes from the inducible promoter in pEpiLac. Up to 300-fold induction can be observed when luciferase is used as a reporter. Inducible expression of the p27KIP1 cyclin-dependent kinase (CDK) inhibitor, the orphan nuclear receptor Nur77 and the angiogenic inducer Cyr61 has also been demonstrated. Expression of the exogenous gene is promptly halted on removal of IPTG. Moreover, the episomal vector can be stably maintained in and easily recovered from MCF7/LAP5 cells. Taken together, this inducible expression system should be applicable for the regulated expression of exogenous genes, especially growth inhibitory or cytotoxic genes, in cells of primate origin.

Heightened Expression of CTCF in Breast Cancer Cells Is Associated with Resistance to Apoptosis

“…16). Construction of the IPTG-inducible episomal vector pEpiLac3 and pEpiLacCTCF (in both orientations) was described previously (17,18). A schematic outline of the antisense construct, pEpiLacCTCFanti, is shown in Fig.…”

CTCF functions as a critical regulator of cell-cycle arrest and death after ligation of the B cell receptor on immature B cells

“…Construction of the IPTGinducible episomal vector pEpiLac3 was described (21). The Ϸ2.7-kb SpeI-SpeI DNA fragment of the mouse CTCF cDNA clone p5.1 (5) was blunt-end ligated in the unique NotI site of the pEpiLac3.…”

“…2 A). The sense and antisense CTCF constructs and the empty pEpiLac3 vector were transfected into the WEHI 231 cells by electroporation and then selected in Hygro by using the technique of Li and Lau (21). The Hygro-resistant cells were single-cell cloned and tested for expression of inducible CTCF by Western blotting and antisense CTCF by Northern blotting after the addition of 8 M IPTG.…”