Methodology article

Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina

Craig Horbinski¹ , Michal K Stachowiak² , Dennis Higgins¹ and Sarah G Finnegan³

¹  Dept. of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New York, USA

²  Molecular and Structural Neurobiology and Gene Therapy Program, Department of Anatomy and Cell Biology, State University of New York at Buffalo, Buffalo, New York USA

³  Dept. of Neurology, State University of New York at Buffalo, Buffalo, New York, USA

author email corresponding author email

BMC Neuroscience 2001, 2:2doi:10.1186/1471-2202-2-2

Published:	16 February 2001

Abstract

Background

Chemical 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.

Results

Optimal 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.

Conclusions

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