Cationic lipasome-mediated DNA transfection in organotypic explant cultures of the ventral mesencephalon

Karl D Murray, A. McQuillin, L. Stewart, C. J. Etheridge, R. G. Cooper, A. D. Miller, H. M D Gurling

Research output: Contribution to journalArticle

21 Scopus citations

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 3β-[N(N'N'-dimethylaminoethane)carbamoyl] cholestrol (DC-Chol) and dioleoyl-L-α-phosphatidylethanolamine (DOPE) could achieve high transfection levels in a neuronal cell line. We therefore wished to assess transfection efficiencies in organotypic cultures from the brain with a reporter plasmid expressing E. coli β-galactosidase in order to mimic an in vivo model. Explant cultures were generated according to the method of Stoppini et al 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 β-galactosidase expression was defected in cells of both neuronal and non-neuronal morphology. Control cultures were exposed to liposome alone and a plasmid that had the β-galactosidase gene insert removed. Only low levels of endogenous β-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.

Original languageEnglish (US)
Pages (from-to)190-197
Number of pages8
JournalGene Therapy
Volume6
Issue number2
DOIs
StatePublished - Feb 1999
Externally publishedYes

Keywords

  • β-galactosidase
  • DC-Chol
  • DOPE
  • Gene therapy
  • Primary culture

ASJC Scopus subject areas

  • Genetics

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    Murray, K. D., McQuillin, A., Stewart, L., Etheridge, C. J., Cooper, R. G., Miller, A. D., & Gurling, H. M. D. (1999). Cationic lipasome-mediated DNA transfection in organotypic explant cultures of the ventral mesencephalon. Gene Therapy, 6(2), 190-197. https://doi.org/10.1038/sj.gt.3300743