title: Polyethylenimine improves the transfection efficiency of primary cultures of post-mitotic rat fetal hypothalamic neurons.
abstract: Analysis of gene regulatory sequences in primary cultures of neurons has been hampered by inefficient transfection of post-mitotic neurons with reporter plasmids. We describe detailed conditions that allowed a significant improvement of transfection efficiency in primary cultures of serum-supplemented rat fetal hypothalamic cells. Transfected cells expressed the green fluorescent protein (GFP) under the control of the strong but non-cell-specific cytomegalo virus (CMV) promoter or under the thyrotropin-releasing hormone (TRH) promoter, to direct expression only in TRH neurons. Using the CMV promoter-GFP plasmid, we tested several commercially available transfection reagents; the best results were obtained with polyethylenimine (PEI) and Lipofectamine 2000. We optimized the transfection procedure with PEI because it rendered more reproducible results. Transfection with PEI was optimal when cells were transfected at a cellular density of 2.9 x 10(6) cells in 35-mm dishes, with 10 microg of DNA, a PEI/DNA ratio of 8.8 and PEI pH of 6.9. Using these conditions, we were able to detect GFP positive neurons after transfecting the TRH promoter-GFP plasmid. GFP positive cells were successfully purified by FACS. This opens the possibility to use transfection of mammalian CNS post-mitotic neurons for new applications including the purification of specific neuronal subtypes.
authors.affiliation: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A.P. 510-3, Cuernavaca, Mor. 62210, Mexico.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Guerra-Crespo
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Charli
authors.forename: Víctor Hugo
authors.initials: VH
authors.lastname: Rosales-García
authors.forename: Gustavo
authors.initials: G
authors.lastname: Pedraza-Alva
authors.forename: Leonor
authors.initials: L
authors.lastname: Pérez-Martínez
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002452:Cell Count; D048429:Cell Size; D002470:Cell Survival; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000378:metabolism; D004622:Embryo, Mammalian; D005260:Female; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods; D049452:Green Fluorescent Proteins; D007031:Hypothalamus / Q000166:cytology* / Q000821:virology; D017403:In Situ Hybridization / Q000379:methods; D008156:Luciferases / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000187:drug effects* / Q000502:physiology / Q000821:virology; D011094:Polyethyleneimine / Q000494:pharmacology*; D011247:Pregnancy; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism; D013997:Time Factors; D014162:Transfection / Q000379:methods*
chemical_list: D008164:Luminescent Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D013973:Thyrotropin-Releasing Hormone; D011094:Polyethyleneimine; D008156:Luciferases