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Design of the Auto-inducible GeneSwitch system

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The previous version of the GeneSwitch® system consisted of two plasmids. pGS1158 expresses the GeneSwitch regulator protein (GLp65.1) under the constitutive control of the CMV immediate-early enhancer/promoter, and pAP1205 is an inducible plasmid with a promoter that contains six copies of the GAL4 DNA binding sites linked to a TATA box which controls expression of a transgene, secreted alkaline phosphatase (SEAP). To lower the extent of unregulated expression associated with the CMV promoter we have constructed a promoter for the GeneSwitch regulator protein that, like the intended target gene, is responsive to mifepristone (MFP). This promoter contains 4 copies of the GAL4 DNA binding sites linked to a minimal thymidine kinase (tk) promoter. The GeneSwitch plasmid with the auto-inducible promoter (pGS1382) differs from pGS1158 in several other aspects. pGS1158 has the SV40 intron and poly(A) site located downstream of the GeneSwitch coding sequence, whereas pGS1382 contains features – a 5’ untranslated region from CMV, identified as UT12, a synthetic intron (termed IVS8) placed within the 5’ UTR, and hGH poly(A) signal, that function in concert to maximize gene expression. In the absence of MFP, the GeneSwitch regulator protein is expressed at low levels from the minimal tk promoter. Upon binding of MFP to the modified ligand-binding domain (LBD) of the human progesterone receptor, the GeneSwitch regulator protein changes conformation to its active state and induces transgene expression by binding to the GAL4 sites in the inducible target plasmid. This activated GeneSwitch regulator can also interact with the GAL4 binding sites linked to the minimal tk promoter to induce further GeneSwitch protein expression. Therefore, in this auto-inducible GeneSwitch® system, both the transgene protein and GeneSwitch protein are induced.