Unusual Properties
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History of the GeneSwitch System

GeneSwitch Regulator Protein, version 1.0
(GS 1.0, GLVP)

The first GeneSwitch® system was constructed by Wang et al., 1994. The design of the chimeric regulator protein was based on the usual properties of the mutant progesterone receptor, hPR-B891, and exploited the modular nature of the functional domains (transactivation, DNA binding, ligand binding) of steroid receptors (Evans, 1988; Picard et al., 1988). The first version GeneSwitch regulator protein (initially termed GLVP) was a chimera of herpes simplex virus VP16 (amino acids 401-476), yeast GAL4 (amino acids 2-93), and the human progesterone receptor (amino acids 640-891). The order of the functional domains in GS 1.0 mimicked that found in the native progesterone receptor. The structure of GS 1.0, including the amino acid sequence of the linker regions, is shown below.

Version 1

MDSQQPDL / VP16 (401-476) / WDL / yGAL4 (2-93) / EFPGVDQ / hPR-LBD D42 (640-891)

VP16 - The amino-terminal region of the native human progesterone receptor, which contains the activation domain, was not sufficient for high levels of transcriptional activation. Thus, the potent transactivation domain from VP16 (amino acids 401-476), a herpes simplex virus type 1 transcription factor (Triezenberg et al., 1988), was employed to ensure efficient transactivation. The VP16 activation domain was placed at the N-terminus of the chimeric protein, just before the GAL4 DNA binding domain.

GAL4 - The progesterone response element (PRE) of progesterone responsive genes is similar, if not identical, to the glucocorticoid and androgen response elements. To prevent the activation of these groups of endogenous hormone responsive genes in target cells, the DNA binding domain of the mutant progesterone receptor was replaced with the amino terminal portion of the yeast transcription factor GAL4 (amino acids 2-93) (Giniger and Ptashne 1988). This portion of the GAL4 protein contains a DNA binding function (amino acids 9-65), a dimerization function (amino acids 65-93), and a nuclear localization signal (amino acids 1-29). The GAL4 DNA binding domain specifically recognizes the GAL4 binding site, a 17 bp DNA sequence which is present in multiple copies in the galactose-regulatable genes of yeast (Giniger et al., 1985), but not in eukaryotic genes. The chimeric GeneSwitch regulator protein, when activated by an antiprogestin drug, should therefore bind only to target genes (i.e. the transgene of interest) that have promoters with GAL4 binding sites.

hPR-LBD - This portion of the progesterone receptor (amino acids 640-891) corresponds to the isolated ligand binding domain with the 42 amino acid C-terminal deletion. As described above, the 42 amino acid deletion causes loss of its ability to bind and become activated by progesterone or other progesterone receptor agonists, but retention of its ability to bind progesterone receptor antagonists, and, unexpectedly, acquisition of an ability to become activated by them. The truncated ligand binding domain was placed at the C-terminus of the chimeric GeneSwitch regulator protein.

In transient transfection studies with plasmids, basal transgene expression is low or undetectable, but induced expression in response to mifepristone is only modest. For example, when cells were co-transfected with plasmids for CMV-GS 1.0 (CMV promoter drives expression of the GS 1.0 protein) and an inducible transgene with a promoter consisting of four GAL4 sites linked to a TATA box, mifepristone at 10-8 M induced transgene expression only 24-fold (Wang et al., 1997a).

In contrast, extremely tight, highly inducible transgene regulation was observed when plasmids for the GeneSwitch® system were integrated into the chromosomal DNA of transgenic mice. In these experiments, the liver-specific transthyretin promoter was used to drive the expression of the GS 1.0 protein and this gene was flanked by chromosomal insulator sequences. The inducible promoter for the transgene (human growth hormone, hGH) contained four GAL4 sites linked to a TATA box. Transgenic mice for the individual genes were generated separately; then, bigenic mice were generated by mating. In the serum of bigenic mice, basal hGH expression was virtually undetectable and administration of mifepristone induced hGH expression over 4 orders of magnitude (Wang et al., 1997a). Mifepristone was administered orally or by intraperitoneal injection at doses of 0.1-0.5 mg/kg body weight. Kinetic analysis of hGH transgene expression revealed that expression peaks at about 12 hours following mifepristone administration and then soon decreases — reaching baseline within 100 hours. The kinetics of abatement of hGH expression correspond to the rate of metabolism and clearance of mifepristone from the body. Readministration of mifepristone reactivated hGH expression — indicating that the target gene can be turned on and off repeatedly by pulsatile administration of mifepristone. Chronic mifepristone treatment of the bigenic mice stimulated a weight gain of 50-60% — thus, demonstrating the ability of the inducible system to cause a noticeable phenotypic change in vivo.