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pFB-ERV: Retroviral Delivery of the Ecdysone Receptor Proteins

with 10 M ponA or an equivalent volume of vehicle. All 24 of the infected lines showed a pon A- dependent induction to some degree (Figure 3). One clone, A610-20 (clone #20, Figure 3) gave an induction of greater than 200-fold in the initial screen. A retest of this cell line gave an induction of approximately 250-fold (Figure 3B ). This clone was produced by infection at an MOI of 0.1, and, therefore, the colony theoretically has a single integrated copy of the receptor expression cassette.

Conclusions

The 200-fold induction seen by transient reporter transfection of the A610-20 receptor line is likely to be an under-representation of the induction profile to be expected in double-stable lines made by selecting stably integrated inducible vectors. This is due to the tight repression that occurs for chromosomally integrated ecdysone-responsive promoters, compared with the relatively high background normally seen in transient transfection assays in which the reporter is free in the nucleus. The stable cell line ER-CHO, which was made using the receptor-expressing plasmid pERV3, shows at best 50-fold induction ratios by transient reporter transfection but the derivative double-stable line consistently gives induction ratios of 700- greater than 1,000-fold.3 Accordingly, we expect that the A610-20 receptor line will give rise to double-stable lines with induction ratios well in excess of 1,000-fold.

Retroviral delivery of the ecdysone receptors should significantly decrease the time and labor required to screen for stable clones that express the receptors at optimal levels, particularly for cell types that are normally difficult to transfect. However, for difficult-to-transfect cell types, delivery of the receptors with the pFB-ERV virus only solves half the problem. Currently, th
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