( change its DNA-binding specificity to...)Versatile Vectors for Ponasterone A- Inducible Control of Gene Expression in,,,Mammalian Cells

change its DNA-binding specificity to that for the glucocorticoid receptor (GR).³ The GR-EcR (GEcR) fusion protein retains the ability to dimerize with RXR and trans-activate in a ponA-dependent manner and is able to recognize and activate reporters containing the synthetic binding site AGTGCA N1 TGTTC (E/GRE). This binding site is extremely unlikely to be recognized by steroid family receptors, which require perfect inverted half-sites, or by natural RXR heterodimer family receptors, which require single nucleotide spacing between half-sites. Finally, the GEcR receptor was further modified by replacing the EcR AD with the more potent VP16 trans-activator to create the receptor protein VgEcR (Figure 1).

Fig.1

Versatile Vectors

Two vectors are required for ponA-inducible expression of the gene of interest: the receptor expression vector pERV3, from which VgEcR and RXR are constitutively expressed, and the ecdysone-inducible vector pEGSH (Figure 2). The pERV3 vector is engineered such that both receptors are expressed from a single mRNA transcribed from the CMV promoter. We accomplished this by placing the internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) upstream of the second (RXR) open reading frame (ORF), which allows high-level internal (cap-independent) initiation of translation of ORFs positioned downstream in an appropriate context.⁴ Expressing both receptors from a single transcript has many advantages. For example, transcription of this expression cassette can be achieved in a wide variety of cell types from a single plasmid, due to the versatility
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