To demonstrate the quality of both the receptor expression and ecdysone-inducible vectors, we performed transient expression assays using the reporter pEGSH-luc in which the coding sequence for the firefly luciferase gene is inserted into the MCS of pEGSH. In these experiments, reporter was cotransfected with various amounts of the pERV3 receptor vector, induced with murA, and assayed for luciferase activity. Figure 3 shows that, in CHO cells, optimal expression is achieved with a relatively small amount of receptor vector (8 ng) to give an induction ratio of over three orders of magnitude. We achieved comparable results for similar experiments carried out in NIH3T3, 293, and CV-1 cells (data not shown).
The best method to engineer double-stable cell lines is to first produce stable receptor-expressing cell lines using the plasmid pERV3 and then screen stable receptor-expressing lines by transient transfection of the inducible reporter pEGSH-luc to find one that mediates the highest level of ponA-dependent trans-activation. Once produced, this line can be used for constructing pEGSH-derived lines. To expedite this process, we have produced three engineered cell lines derived from CHO, NIH3T3, and 293 cells, respectively, in which optimal levels of the receptors are stably expressed. These three lines show high-level inducer-dependent activation in transient expression assays using the pEGSH-luc reporter (Figure 4).
The stable receptor line ER-CHO (Figure
4A ) was stably transfected with the plasmid pEGSH-luc, and hygromycin-