title: Cationic liposome-mediated DNA transfection in organotypic explant cultures of the ventral mesencephalon.
abstract: We have examined the potential of cationic liposomes as a tool for approaches to gene therapy in the CNS. Our previous work has shown that cationic liposomes formulated from 3 beta-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol (DC-Chol) and dioleoyl-L-alpha-phosphatidylethanolamine (DOPE) could achieve high transfection levels in a neuronal cell line (McQuillin et al. Neuroreport 1997; 8: 1481-1484). We therefore wished to assess transfection efficiencies in organotypic cultures from the brain with a reporter plasmid expressing E. coli beta-galactosidase in order to mimic an in vivo model. Explant cultures were generated according to the method of Stoppini et al (J Neurosci Meth 1991; 37: 173-182) with slight modifications. Brain slices were maintained on transparent porous membranes and were observed to maintain their intrinsic connectivity and cytoarchitecture to a large degree over periods of up to 6 weeks in culture. CNS tissue was obtained from rats at birth or 5 days after birth. After transfection beta-galactosidase expression was detected in cells of both neuronal and non-neuronal morphology. Control cultures were exposed to liposome alone and a plasmid that had the beta-galactosidase gene insert removed. Only low levels of endogenous beta-galactosidase reactivity were seen in these control cultures. DC-Chol/DOPE-mediated transfection was confirmed using a RT-PCR protocol capable of differentiating between untranscribed plasmid DNA and RNA generated from the transfected vector. These results suggest that cationic liposomes, particularly DC-Chol/DOPE liposomes, will be useful as delivery agents for gene transfer to CNS cells in vitro and possibly in vivo.
authors.affiliation: Department of Psychiatry and Behavioural Sciences, University College London Medical School, Windeyer Institute of Medical Sciences, UK.
authors.forename: K D
authors.initials: KD
authors.lastname: Murray
authors.forename: A
authors.initials: A
authors.lastname: McQuillin
authors.forename: L
authors.initials: L
authors.lastname: Stewart
authors.forename: C J
authors.initials: CJ
authors.lastname: Etheridge
authors.forename: R G
authors.initials: RG
authors.lastname: Cooper
authors.forename: A D
authors.initials: AD
authors.lastname: Miller
authors.forename: H M
authors.initials: HM
authors.lastname: Gurling
mesh_terms: D000818:Animals; D002493:Central Nervous System Diseases / Q000628:therapy*; D002784:Cholesterol / Q000031:analogs & derivatives; D046508:Culture Techniques; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D008081:Liposomes; D008636:Mesencephalon; D010714:Phosphatidylethanolamines; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D008081:Liposomes; D010714:Phosphatidylethanolamines; C070648:3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol; C020888:1,2-dielaidoylphosphatidylethanolamine; D002784:Cholesterol; D001616:beta-Galactosidase