Polyethyleneimine-mediated transfection of cultured postmitotic neurons from rat sympathetic ganglia and adult human retina.
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 beta-galactosidase or enhanced green fluorescence protein (EGFP) using PEI.


RESULTS
Optimal transfection efficiency was observed with 1 microg/ml of plasmid DNA and 5 microg/ml PEI. Expression of beta-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 approximately 9% of the neurons expressing beta-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.
Horbinski
C
C
Dept. of Pharmacology and Toxicology, State University of New York at Buffalo, Buffalo, New York, USA. horbinsk@acsu.buffalo.edu
Stachowiak
M K
MK
Higgins
D
D
Finnegan
S G
SG
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005728:Ganglia, Sympathetic / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008938:Mitosis; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011094:Polyethyleneimine / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D014162:Transfection / Q000379:methods*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D008164:Luminescent Proteins; D011094:Polyethyleneimine; D004247:DNA; D001616:beta-Galactosidase