title: High-efficiency transfection of mammalian neurons via nucleofection.
abstract: Transfection of foreign DNA is widely used to study gene function. However, despite the development of numerous methods, the transfer of DNA into postmitotic cells, such as neurons, remains unsatisfactory with regard to either transfection efficiency or cytotoxicity. Nucleofection overcomes these limitations. Direct electroporation of expression plasmids or oligonucleotides into the nucleus ensures both good cell viability and consistently high transfection rates. This allows biochemical analyses of transfected neurons, for example, western blot analyses of protein levels after RNA interference (RNAi) knockdown or microRNA transfection. We provide comprehensive protocols for performing nucleofection with high efficiency on primary neurons. The focus is on the recently developed 96-well shuttle system, which allows the simultaneous testing of up to 96 different plasmids or experimental conditions. Using this system, reproducible high-throughput expression of various transgenes is now feasible on primary neurons, for example large-scale RNAi analyses to downregulate gene expression. The protocol typically takes between 2 and 3 h.
authors.affiliation: Division of Neuronal Cell Biology, Center for Brain Research, Medical University of Vienna, A-1090 Vienna, Austria.
authors.forename: Manuel
authors.initials: M
authors.lastname: Zeitelhofer
authors.forename: John P
authors.initials: JP
authors.lastname: Vessey
authors.forename: Yunli
authors.initials: Y
authors.lastname: Xie
authors.forename: Fabian
authors.initials: F
authors.lastname: Tübing
authors.forename: Sabine
authors.initials: S
authors.lastname: Thomas
authors.forename: Michael
authors.initials: M
authors.lastname: Kiebler
authors.forename: Ralf
authors.initials: R
authors.lastname: Dahm
mesh_terms: D000818:Animals; D019470:Biolistics; D002130:Calcium Phosphates; D004247:DNA / Q000235:genetics; D005821:Genetic Techniques; D007202:Indicators and Reagents; D016086:Lentivirus; D008322:Mammals; D008938:Mitosis; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D034622:RNA Interference; D012190:Retroviridae; D014162:Transfection / Q000379:methods*
chemical_list: D002130:Calcium Phosphates; D007202:Indicators and Reagents; D004247:DNA; C020243:calcium phosphate