Adeno-associated virus mediated gene transfer into primary rat brain neuronal and glial cultures: enhancement with the pH-sensitive surfactant dodecyl 2-(1'-imidazolyl) propionate.
This study evaluated the effects of a novel, pH-sensitive surfactant, dodecyl 2-(1'-imidazolyl) propionate (DIP), on cationic lipid mediated transfection in primary rat brain neuronal and glial cultures. The cationic lipid complex DOTAP/DOPE (1, 2-dioleoyl-3-trimethylammonium propionate and dioleoyl phosphatidylethanolamine, respectively) was added over a range of concentrations (0-120 microg/ml) with DNA concentration kept constant (1.6 microg/ml). The neuron-specific enolase (NSE) and cytomegalovirus (CMV) promoters were found to drive green fluorescent protein (GFP) expression in neuron-enriched and glial cultures, respectively, using adeno-associated virus (AAV) derived constructs. NSE-driven GFP expression was not observed in glial cultures. Addition of DOTAP/DOPE increased transfection efficiency over a wide range of lipid concentrations (5-50 microg/ml) keeping DNA concentration constant (1.6 microg/ml). Addition of DIP to the lipid/DNA complex increased maximum transfection efficiencies in glial and neuronal cultures 2-3-fold. Transfection efficiencies were at their maximum with a similar total lipid concentration (50 microg/ml) in both cell-types in the presence of DIP. Neuronal cultures were more sensitive than glia to the toxic actions of DOTAP/DOPE, with or without DIP. These results indicate that AAV-mediated gene-transfer to neurons and glia can be facilitated by addition of a pH-sensitive surfactant to cationic liposome/DNA complexes and that endosomal escape could be a limiting factor in transgene expression.
Wang
S
S
Department of Pharmacology, University of Florida, Gainesville 32610, USA.
Bui
V
V
Hughes
J A
JA
King
M A
MA
Meyer
E M
EM
D000818:Animals; D001921:Brain / Q000166:cytology / Q000502:physiology*; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology; D015870:Gene Expression / Q000502:physiology; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics; D009457:Neuroglia / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D010714:Phosphatidylethanolamines / Q000494:pharmacology; D010751:Phosphopyruvate Hydratase / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000502:physiology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D051381:Rats; D013501:Surface-Active Agents / Q000494:pharmacology
D005229:Fatty Acids, Monounsaturated; D007093:Imidazoles; D008164:Luminescent Proteins; C100541:N-dodecyl-2-imidazole propionate; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D013501:Surface-Active Agents; D049452:Green Fluorescent Proteins; C094877:dioleoyl phosphatidylethanolamine; D010751:Phosphopyruvate Hydratase; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane