Jan 23 2019
A research team from KAIST has come up with a noninvasive light-sensitive photoactivatable recombinase that can be used for in vivo genetic manipulation.
This is a schematic view of the research. (Image credit: KAIST)
Photoactivatable Flp recombinase is highly light-sensitive, making it best suited for controlling genetic manipulation in deep regions of the mouse brain by using a noninvasive light-emitting diode for illumination. Professor Won Do Heo and his colleagues developed this easy-to-use optogenetic module, which will offer an expandable and side-effect-free genetic manipulation tool for neuroscience research.
Spatiotemporal control of the expression of genes has been commended as a useful approach for recognizing the functions of genes that have complex neural circuits. Robust and extremely advanced technologies that facilitate specific labeling and fast genetic modification in live animals are needed for the investigation of complex brain functions. Several strategies have been developed for controlling the protein activity or gene expression in a spatiotemporal way using hormones, light, peptides, and small molecules to manipulate intact circuits or functions.
Of these, chemically inducible, recombination-employing systems are the most usually employed in vivo gene-modification systems. Other strategies include conditional or selective Cre-activation systems within subsets of dual-promoter-driven intersectional populations of cells or green fluorescent protein-expressing cells.
Yet, the drawbacks of these techniques are that they need considerable effort and time to establish knock-in mouse lines and face constraints on spatiotemporal control, which is dependent on a limited set of available transgenic mouse resources and genetic promoters.
In spite of these constraints, optogenetic strategies enable the activity of genetically defined mouse brain neurons to be controlled with high spatiotemporal resolution. However, it has been difficult to develop an optogenetic module for gene-manipulation with the ability to reveal the spatiotemporal functions of particular target genes in the mouse brain.
The researchers reported about the photoactivatable Flp recombinase in the study published in Nature Communication on January 18th, 2019, by looking out for split sites of Flp recombinase that were earlier not identified, with the potential of reconstitution to be active. They validated the efficient and highly light-sensitive performance of photoactivatable Flp recombinase through accurate light targeting by demonstrating transgene expression within anatomically confined regions of the mouse brain.
The idea of local genetic labeling reported in this study proposes a new strategy for genetically recognizing subpopulations of cells defined by the spatial and temporal characteristics of light delivery. Until now, developing an optogenetic module for gene manipulation with the potential to reveal the spatiotemporal functions of particular target genes in the mouse brain has not been feasible and such a light-inducible Flp system has been out of reach. Hence, the researchers made efforts to create a photoactivatable Flp recombinase that completely exploits the high spatiotemporal control provided by light stimulation.
This activation using noninvasive light illumination deep within the brain is beneficial since it prevents side effects mediated by chemical or optic fiber implantation, for example, off-target cytotoxicity or physical lesions that might have an impact on animal physiology or behaviors. The method offers expandable functionalities for transgene expression systems during in vivo Flp recombinase activity, by outlining a viral vector for minimal leaky expression affected by viral nascent promoters.
The researchers showed the functionality of PA-Flp as a noninvasive in vivo optogenetic manipulation tool that can be used in the mouse brain, even applicable for deep brain structures since it has the ability to reach the medial septum or hippocampus with the help of external LED light illumination.
The study is the outcome of five years of research performed by Professor Heo, who has headed the optogenetics and bio-imaging fields by creating his own optogenetics and bio-imaging technologies.
It will be a great advantage to control specific gene expression desired by LEDs with little physical and chemical stimulation that can affect the physiological phenomenon in living animals.
Won Do Heo, Professor, KAIST.