title: An Optimized Calcium-Phosphate Transfection Method for Characterizing Genetically Encoded Tools in Primary Neurons.
abstract: In order to characterize genetically encoded tools under the most relevant conditions, the constructs need to be expressed in the cell type in which they will be used. This is a major hurdle in developing optogenetic tools for neuronal cells, due to the difficulty of gene transfer to these cells. Several protocols have been developed for transfecting neurons, focusing on improved transfection efficiency. However, obtaining healthy cells is as important. We monitored transfected cell health by measuring electrophysiological parameters, and used them as a guideline to optimize transfection. Here we describe an optimized transfection protocol that achieves reasonably high efficiency (10-20 %) with no discernable impact on cell health, as characterized by electrophysiology.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT, USA.
authors.forename: Shiyao
authors.initials: S
authors.lastname: Wang
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Connecticut, Storrs, CT, USA. cho@engr.uconn.edu.|Institute for Systems Genomics, University of Connecticut, Storrs, CT, USA. cho@engr.uconn.edu.
authors.forename: Yong Ku
authors.initials: YK
authors.lastname: Cho
mesh_terms: D000818:Animals; D002130:Calcium Phosphates / Q000378:metabolism*; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D055724:Electrophysiological Phenomena; D006624:Hippocampus / Q000166:cytology; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D062308:Optogenetics / Q000379:methods; D014162:Transfection / Q000379:methods*
chemical_list: D002130:Calcium Phosphates; C020243:calcium phosphate
keywords: Dissociated culture; Electrophysiology; Optogenetics; Primary neurons; Transfection