Modified GFAP promoter auto-regulates tet-activator expression for increased transactivation and reduced tTA-associated toxicity

Abstract

Transactivator tTA is a necessary component of the tetracycline-regulated inducible gene system. While several transgenic animals have been described that express tTA in the central nervous system (CNS), their tTA levels are often limited, presumably due to toxic effects. We evaluated methods for auto-regulating tTA levels in astrocytes by modifying the transgenic promoter human GFAP (hGFAP). The hGFAP promoter carrying a single copy of the tet-operon in place of a native enhancer element (GFAPtetO1) drove expression of tTA at low levels during un-stimulated, basal condition. However the same promoter auto-induced expression of tTA to significant levels after tetracycline withdrawal. Glial cell-specificity of the promoter remained uncompromised during both basal and induced conditions. Transgenic rats were developed using the auto-inducible GFAPtetO1 promoter that expressed tTA mRNA to high levels in the brain. Expression was widespread within the CNS but enriched in astrocyte-rich regions including the cerebellum. Primary cerebellar astrocytes from GFAPtetO1 rats transfected with 07LacZ produced substantially greater inducibility of reporter gene compared to GFAP-tTA transgenic rats. Finally, GFAPtetO1 rats exhibited severe motor/gait deficit when bred to homozygosity. This phenotype was attributable to developmental abnormalities of the cerebellum and was completely abrogated by doxycycline administration. These results suggest that developmental toxicity resulting from tTA expression can be circumvented and tTA transgenics with high transactivation potential can be developed using the auto-activation strategy. Promoter modification presented here may be useful in developing highly inducible transgenic strategies without loss in tissue-specificity.