title: Fast targeted gene transfection and optogenetic modification of single neurons using femtosecond laser irradiation.
abstract: A prevailing problem in neuroscience is the fast and targeted delivery of DNA into selected neurons. The development of an appropriate methodology would enable the transfection of multiple genes into the same cell or different genes into different neighboring cells as well as rapid cell selective functionalization of neurons. Here, we show that optimized femtosecond optical transfection fulfills these requirements. We also demonstrate successful optical transfection of channelrhodopsin-2 in single selected neurons. We extend the functionality of this technique for wider uptake by neuroscientists by using fast three-dimensional laser beam steering enabling an image-guided "point-and-transfect" user-friendly transfection of selected cells. A sub-second transfection timescale per cell makes this method more rapid by at least two orders of magnitude when compared to alternative single-cell transfection techniques. This novel technology provides the ability to carry out large-scale cell selective genetic studies on neuronal ensembles and perform rapid genetic programming of neural circuits.
authors.affiliation: 1] University of St Andrews, School of Biology, St Andrews, UK [2] SUPA, University of St Andrews, School of Physics & Astronomy, St Andrews, UK [3] SULSA, Scottish Universities Life Sciences Alliance.
authors.forename: Maciej
authors.initials: M
authors.lastname: Antkowiak
authors.forename: Maria Leilani
authors.initials: ML
authors.lastname: Torres-Mapa
authors.forename: Emily C
authors.initials: EC
authors.lastname: Witts
authors.forename: Gareth B
authors.initials: GB
authors.lastname: Miles
authors.forename: Kishan
authors.initials: K
authors.lastname: Dholakia
authors.forename: Frank J
authors.initials: FJ
authors.lastname: Gunn-Moore
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002478:Cells, Cultured; D000075402:Channelrhodopsins; D005260:Female; D007834:Lasers; D008164:Luminescent Proteins / Q000235:genetics; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D062308:Optogenetics; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D013997:Time Factors; D014162:Transfection; D014743:Videotape Recording
chemical_list: D001426:Bacterial Proteins; D000075402:Channelrhodopsins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria