Abstract
Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Here, we develop a highly light-sensitive and efficient photoactivatable Flp recombinase (PA-Flp) that is suitable for genetic manipulation in vivo. The highly light-sensitive property of PA-Flp is ideal for activation in deep mouse brain regions by illumination with a noninvasive light-emitting diode. In addition, PA-Flp can be extended to the Cre-lox system through a viral vector as Flp-dependent Cre expression platform, thereby activating both Flp and Cre. Finally, we demonstrate that PA-Flp–dependent, Cre-mediated Cav3.1 silencing in the medial septum increases object-exploration behavior in mice. Thus, PA-Flp is a noninvasive, highly efficient, and easy-to-use optogenetic module that offers a side-effect-free and expandable genetic manipulation tool for neuroscience research.
Highlights
Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits
Two optogenetic modules of photoactivatable-Cre recombinase (PA-Cre) have been developed using split-Cre components, one in which each component is fused to CRY2 and CIB112–14, and the other in which they are fused to positive Magnet and negative Magnet[15]
In designing photoactivatable Flp recombinase (PA-Flp), we employed a strategy in which Flpo was split into two pieces that reassemble in response to a light stimulus (Fig. 1a)
Summary
Spatiotemporal control of gene expression or labeling is a valuable strategy for identifying functions of genes within complex neural circuits. Other approaches include selective or conditional Cre-activation systems within subsets of green fluorescent protein (GFP)-expressing cells (Cre-DOG)[10] or dualpromoter-driven intersectional populations of cells[11] These methods are limited by the considerable time and effort required to establish knock-in mouse lines and by constraints on spatiotemporal control, which relies on a limited set of available genetic promoters and transgenic mouse resources. Two optogenetic modules of photoactivatable-Cre recombinase (PA-Cre) have been developed using split-Cre components, one in which each component is fused to CRY2 and CIB112–14, and the other in which they are fused to positive Magnet (pMag) and negative Magnet (nMag)[15] In both cases, illumination with blue light induces heterodimerization. We engineer a Flpdependent Cre driver as a module without leaky Cre expression in viral vector system, showing noninvasive light-dependent, Cre-mediated Cav3.1 gene silencing in MS neurons, which results in increased objective exploration behavior
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