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I. Richard, M. Ader, V. Sytnyk, A. Dityatev, G. Richard, M. Schachner, U. Bartsch; Efficient Transfection of Neural Stem Cells by Electroporation . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2338.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Transplantation of neural stem cells (NSCs) is a potential tool to replace degenerated cell types in the nervous system. An efficient technique to genetically modify these cells might be of use to optimize, for instance, the integration, differentiation and/or survival of grafted NSCs in the host tissue. In addition, transfected NSCs might be used as cellular vehicles to transfer therapeutic gene products to the diseased nervous system. Here, we describe a novel electroporation technique which allows the efficient transfection of NSCs from the spinal cord of mouse embryos. Methods: NSCs were isolated from the spinal cord of wild-type, L1-deficient or enhanced green fluorescent protein- (EGFP) transgenic mouse embryos. Wild-type cells were transfected with the reporter gene EGFP and L1-deficient or EGFP-transgenic cells with the cell recognition molecule L1 using the NucleofectorTM Device and Kit (Amaxa Biosystems, Köln, Germany). The transfection efficacy was determined one day after transfection. Expression of EGFP and L1 was also monitored in differentiating NSC cultures in vitro and after intraretinal transplantation in vivo. Results: Quantitative analysis of cultures one day after transfection revealed that about 70% of wild-type cells and L1-deficient cells expressed EGFP and L1, respectively. When cultures were induced to differentiate in vitro, EGFP-positive and L1-immunoreactive cells were detectable for up to 3.5 weeks, the longest time interval investigated. Finally, when EGFP- or L1-transfected NSCs were grafted into the retina of adult wild-type or L1-deficient mice, EGFP- and L1-positive cells could be detected in the host tissue for up to 2 and 3.5 weeks, respectively, the longest post-transplantation intervals investigated. Conclusions: Results indicate that this novel method is a powerful non-viral transfection technique to genetically modify NSCs to combine cell therapy with gene therapy.
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