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Noninvasive Optical Activation of Flp Recombinase in Deep Mouse Brain Regions

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 an Flp-dependent Cre expression platform, thereby activating both Flp and Cre.

As a research project of the department of biological sciences at KAIST and the center for cognition and sociality in IBS, Professor Won Do Heo and his colleague developed photoactivatable Flp recombinase (PA-Flp), which has high sensitivity in deep brain regions of mice. Investigating the function of genes or circuits in diverse brain regions requires molecular tools capable of precise spatiotemporal genetic manipulation. Among the tools most widely used to date for neuroscience research applications are inducible Cre recombinase knock-in mouse lines in which Cre expression is driven by tissue- or cell type-specific promoters. However, inducible Flp recombinase function has rarely been established in vivo. The emerging need for sophisticated gene-modification tools for interrogating complex neural networks can be expected to enhance interest in inducible Flp systems for combinatorial or simultaneous use with Cre.

Heo’s group developed a photo-activatable Flp recombinase (PA-Flp) that is highly sensitive to activation by blue light and is applicable for use in the mouse brain. To develop PA-Flp, they used blue-light–activated hetero-interaction modules called a positive magnet (pMagH) and a negative magnet (nMagH), each fused to split Flp fragments that are designed to be reconstituted and activated upon light illumination. Also, they validated the ability of PA-Flp to act on exogenous or endogenous Flp reporters in vivo in the mouse brain. They demonstrated the light-dependent efficacy of PA-Flp, delivered via plasmid electroporation or rAAV, in the mouse brain. The high light sensitivity of PA-Flp further enabled us to verify the concept of genetic labeling of anatomically restricted areas (~200-μm scale) with minimal light scattering. Surprisingly, PA-Flp could be activated by noninvasive LED illumination for as little as 30 seconds, reaching the hippocampus or medial septum (MS)—both deep brain structures—even without removing the skull or skin.

To further refine the process, the team established a double-check control system that blocks translation of transcripts mediated by nascent viral promoters, thereby providing stringent leak-free transgene expression in vivo. As a representative application of this system, team demonstrated that a Leak-Free Flp-dependent Cre driver (LF-FdCd), in which a neuron-specific promoter drives native Cre expression, produces a pattern that mirrors the subsets of cell populations that feature Flp activity.

Hyunjin Jung, Prof. Won Do Heo
2019 KI Newsletter

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