Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retinaReportar como inadecuado




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BMC Neuroscience

, 2:2

First Online: 16 February 2001Received: 14 December 2000Accepted: 16 February 2001

Abstract

BackgroundChemical methods of transfection that have proven successful with cell lines often do not work with primary cultures of neurons. Recent data, however, suggest that linear polymers of the cation polyethyleneimine PEI can facilitate the uptake of nucleic acids by neurons. Consequently, we examined the ability of a commercial PEI preparation to allow the introduction of foreign genes into postmitotic mammalian neurons. Sympathetic neurons were obtained from perinatal rat pups and maintained for 5 days in vitro in the absence of nonneuronal cells. Cultures were then transfected with varying amounts of a plasmid encoding either E. coli β-galactosidase or enhanced green fluorescence protein EGFP using PEI.

ResultsOptimal transfection efficiency was observed with 1 μg-ml of plasmid DNA and 5 μg-ml PEI. Expression of β-galactosidase was both rapid and stable, beginning within 6 hours and lasting for at least 21 days. A maximum yield was obtained within 72 hours with ∼ 9% of the neurons expressing β-galactosidase, as assessed by both histochemistry and antibody staining. Cotransfection of two plasmids encoding reporter genes was achieved. Postmitotic neurons from adult human retinal cultures also demonstrated an ability to take up and express foreign DNA using PEI as a vector.

ConclusionsThese data suggest that PEI is a useful agent for the stable expression of plasmid-encoded genes in neuronal cultures.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2202-2-2 contains supplementary material, which is available to authorized users.

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Autor: Craig Horbinski - Michal K Stachowiak - Dennis Higgins - Sarah G Finnegan

Fuente: https://link.springer.com/







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