In-tube transfection improves the efficiency of gene transfer in primary neuronal cultures.
To facilitate genetic studies in primary neurons, we analyzed the efficiency of cationic lipid-mediated plasmid DNA transfection using adherent and acutely dissociated neuronal suspensions derived from embryonic mouse cortical tissue. Compared to transfections using adherent cultures, the in-tube procedure enhanced the delivery of a GFP reporter plasmid between four- to eightfold depending on the age of the harvested embryo. The procedure required relatively brief complex incubation times, and supported the transfection of cells expressing the neuronal markers NeuN and TuJ1 with improved uniformity in transfection events across the well surface. To demonstrate the utility of this approach in studying the genetic mechanisms controlling neuron development, we provide data regarding the role of the bZIP transcription factor c/EBP-beta in regulating neurite outgrowth. It is anticipated that this in vitro protocol will facilitate the identification of novel genes involved in both developmental and disease-relevant signaling pathways.
Halterman
Marc W
MW
Department of Neurology, University of Rochester Medical Center, Rochester, NY 14642, United States. marc_halterman@urmc.rochester.edu <marc_halterman@urmc.rochester.edu>
Giuliano
Rita
R
Dejesus
Chris
C
Schor
Nina F
NF
D000818:Animals; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008055:Lipids / Q000737:chemistry / Q000494:pharmacology; D051379:Mice; D008967:Molecular Biology / Q000295:instrumentation / Q000379:methods*; D016501:Neurites / Q000378:metabolism / Q000648:ultrastructure; D055495:Neurogenesis / Q000235:genetics; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D010957:Plasmids / Q000235:genetics; D011107:Polylysine / Q000494:pharmacology; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D014162:Transfection / Q000295:instrumentation / Q000379:methods*
D022782:CCAAT-Enhancer-Binding Protein-beta; D008055:Lipids; D049452:Green Fluorescent Proteins; D011107:Polylysine