Lipid-mediated gene transfer into primary neurons using FuGene: comparison to C6 glioma cells and primary glia.
Gene transfer into cells of CNS origin is an important tool to counteract neurodegeneration by introducing, e.g., neuroprotective molecules. Although viral gene transfer reveals the highest gene transfer efficiency, liposome-mediated gene transfer seems to become an attractive alternative. In this study we investigated the lipid-mediated gene transfer into primary neurons in vitro by using the novel nonliposomal lipid FuGene and compared it to primary glia and malignant C6 glioma cells. FuGene-mediated gene transfer was useful to transfer the reporter gene beta-galactosidase into C6 glioma cells, primary glia, and primary neurons. Lipofection was highly dependent on the surface bottom and did not result in good efficiencies when using glass compared to plastic dishes. Comparing to a standard lipofection (1 x 8 h), lipofection on 3 consecutive days for 6 h each ("boosting") markedly increased the gene transfer efficiency in primary glia (up to sevenfold) and in primary neurons (up to sixfold). The use of endotoxin-free DNA only slightly increased the transfection efficiency. Immunohistochemistry demonstrated MAP-2-positive neurons (up to 1614 neurons/16-mm well; 2.4% of total neurons) as well as TH-positive neurons (up to 48 neurons/16-mm well; 12.7% of TH neurons) expressing beta-galactosidase. We conclude that FuGene-mediated gene transfer is an attractive alternative to introduce genes of interest into cells of glial and neuronal origin; however, this technique lacks sufficient gene transfer efficiency.
Wiesenhofer
B
B
Laboratory of Psychiatry, University Hospital Innsbruck, Austria.
Humpel
C
C
D000818:Animals; D002411:Cation Exchange Resins / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media; D004247:DNA / Q000378:metabolism; D005069:Evaluation Studies as Topic; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000235:genetics; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D005910:Glioma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D007150:Immunohistochemistry; D050356:Lipid Metabolism; D008055:Lipids / Q000494:pharmacology*; D008081:Liposomes / Q000008:administration & dosage / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D014162:Transfection / Q000187:drug effects / Q000379:methods; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D002411:Cation Exchange Resins; D003470:Culture Media; C411955:FuGene; D005904:Glial Fibrillary Acidic Protein; D008055:Lipids; C086724:Lipofectamine; D008081:Liposomes; D008869:Microtubule-Associated Proteins; D004247:DNA; D014446:Tyrosine 3-Monooxygenase; D001616:beta-Galactosidase