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.
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Acknowledgements
This work was funded by the University of Connecticut and the Brain and Behavior Research Foundation (NARSAD Young Investigator grant). Shiyao Wang was partially supported by the outstanding scholars program of the UConn graduate school.
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Wang, S., Cho, Y.K. (2016). An Optimized Calcium-Phosphate Transfection Method for Characterizing Genetically Encoded Tools in Primary Neurons. In: Kianianmomeni, A. (eds) Optogenetics. Methods in Molecular Biology, vol 1408. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3512-3_16
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DOI: https://doi.org/10.1007/978-1-4939-3512-3_16
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-3512-3
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