Rapid TetOn-mediated gene expression in neurons across the lifespan with uTTOP

Abstract

Conditional expression of genes of interest is essential for interrogation of cellular development and function. Although tools exist for conditional gene expression, techniques for rapid-onset, temporally precise expression are lacking. The doxycyclineinducible TetOn expression system allows for this in numerous organ systems, however, transgenic TetOn expression cassettes become silenced in the nervous system during postnatal development. Here, we circumvent this silencing with uTTOP:in uteroelectroporation of Transposable TetOn Plasmids. When electroporated as transposable elements that integrate into the genome, the TetOn system allowed for robust DOX-dependent induction of expression across the postnatal lifespan of the mouse. We demonstrated induction in neurons of sensorimotor and retrospleninal cortex, hippocampus and the olfactory bulb. Latency to peak induction was ≤12 hours, a several fold increase in induction kinetics over existing methodology forin vivoconditional expression. To demonstrate the utility of uTTOP, we induced ectopic expression of Sonic hedgehog in adult mouse layer 2/3 cortical neurons, demonstrating that its expression can diversify expression of Kir4.1 in surrounding astrocytes. The rapid induction kinetics of uTTOP allowed us to show that Kir4.1 upregulation significantly lags onset of Shh expression by ∼2 days, a difference in expression time course that is likely not resolvable with current methods. Together, these data demonstrate that uTTOP is a powerful and flexible system for conditional gene expression in multiple brain areas across the mouse lifespan.