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pFB-ERV: Retroviral Delivery of the Ecdysone Receptor Proteins

e system has a number of advantages over alternative systems. Firstly, the lipophilic nature and short in vivo half-life of the ecdysone analog ponasterone A (ponA) allow efficient penetrance into all tissues including brain, resulting in rapid and potent inductions and rapid clearance. Secondly, ecdysteroids are not known, nor are they expected, to affect mammalian physiology in any measurable way. Thirdly, the heterodimeric ponA responsive receptor and receptor DNA recognition element have been genetically altered such that trans-activation of endogenous genes by the ecdysone receptor, or of the ponA-responsive expression cassette by endogenuos transcription factors, is extremely unlikely.

In addition, it has been found that in the absence of inducer the heterodimer remains bound at the promoter in a complex with corepressors and histone deacetylase, and is, thus, tightly repressed until ligand binding, at which time high-level transcriptional activation occurs (i.e., the heterodimer is converted from a tight repressor to a trans-activator). Using the Complete Control plasmid-based system, induction ratios of greater than 1,000-fold have been achieved in both transient transfections and in stable cell lines.3

Using plasmid-based vectors for controlled gene expression is limiting because many cell types that are of academic, industrial, or clinical interest are difficult or virtually impossible to transfect using current transfection methods. In particular, primary human cell lines and lymphocytes are best transduced using viral delivery systems. The most popular and user-friendly of these are the retroviral vectors.4,5 Infection with retroviruses often yields transduction efficiencies close to 100%, and the proviral copy number can be easily controlled by varying the multiplicity of infection (MOI). This latter feature is particularly important for inducible systems for which l
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