Effect of ethanol on lipid-mediated transfection of primary cortical neurons.
Successful introduction of nucleic acids into mammalian neurons has revolutionized the analyses of gene regulation and cellular function. Various methods, including viral infection, have been developed to introduce plasmid DNA into primary neuronal cultures. However, transfection of primary cultures of neurons using the calcium phosphate precipitation method and electroporation have been comparatively inefficient. In this paper, we describe a method to successfully transfect cultured fetal cortical neurons using a cationic lipid reagent, lipofectamine. Cells were cultured in the absence and presence of 50 mM ethanol. To monitor transfection of neurons, we employed three mammalian expression vectors containing Renilla luciferase and/or firefly luciferase, or the beta-galactosidase reporter gene. Fetal cortical neurons were isolated and cultured in the absence or presence of 50 mM ethanol, for two days. On day 3, neurons were washed, fed with serum-free medium, and transfected with the DNA-lipofectamine complex. After two hours, cells were washed, fed complete medium lacking or containing 50 mM ethanol and cultured for two additional days with a change of medium after 24 h. Cultures were terminated 48 h after transfection. Cells were either stained for beta-galactosidase activity using X-gal or lysed to prepare cell extracts to assay for luciferase activity using a luminometer. When neurons were cotransfected, Renilla luciferase was used as an internal control to normalize the expression of the firefly luciferase reporter gene. Analysis of results showed that expression of the reporter gene, firefly luciferase, was approximately 2.5 times greater in ethanol treated neuronal cultures than for neurons cultured in the absence of ethanol. An increased number of neurons expressing beta-galactosidase was also observed in ethanol-treated neurons. These data suggest that perhaps ethanol treatment of fetal cortical neurons improved the DNA uptake and/or increased the expression of the reporter genes.
Anji
Antje
A
Department of Anatomy and Physiology, CVM, Kansas State University, Manhattan 66506, USA.
Shaik
Kamran A
KA
Kumari
Meena
M
D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000502:physiology*; D004247:DNA / Q000378:metabolism; D000431:Ethanol / Q000494:pharmacology*; D005260:Female; D005333:Fetus / Q000033:anatomy & histology / Q000502:physiology; D017930:Genes, Reporter; D005822:Genetic Vectors; D050356:Lipid Metabolism; D008055:Lipids; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D011247:Pregnancy; D014162:Transfection / Q000379:methods*
D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D000431:Ethanol; D004247:DNA