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

Gene Therapy volume 6, pages 190–197 (1999)Cite this article

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] cholesterol (DC-Chol) and dioleoyl-L-α-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 β-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 con- nectivity 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 detected 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.

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Authors and Affiliations

  1. Department of Psychiatry and Behavioural Sciences, University College London Medical School, Windeyer Institute of Medical Sciences, London, UK

    K D Murray, A McQuillin & H M D Gurling

  2. Department of Chemistry, Imperial College of Science, Technology and Medicine, London, UK

    L Stewart, C J Etheridge, R G Cooper & A D Miller

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  1. K D Murray
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  2. A McQuillin
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  3. L Stewart
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  4. C J Etheridge
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  6. A D Miller
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Murray, K., McQuillin, A., Stewart, L. et al. Cationic liposome-mediated DNA transfection in organotypic explant cultures of the ventral mesencephalon. Gene Ther 6, 190–197 (1999). https://doi.org/10.1038/sj.gt.3300743

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