Efficient gene delivery to primary neuron cultures using a synthetic peptide vector system.
A bi-functional, 31 amino acid synthetic peptide (polylysine-molossin) was evaluated for gene delivery to primary cultures of rat cerebral cortex neurons. Polylysine-molossin consists of an amino terminal domain of 16 lysines for electrostatic binding of DNA, and a 15 amino acid, integrin-binding domain at the carboxyl terminal. High levels of gene delivery were obtained with 20-30 microM chloroquine, with a synthetic fusogenic peptide at an optimal DNA:polylysine-molossin:fusogenic peptide w/w ratio of 1:3:0.2, and with the addition of low concentrations of Lipofectamine 2000 at an optimal DNA:polylysine-molossin:Lipofectamine 2000 w/w ratio of 1:3:0.5. With the best combination, >30% of neurons strongly expressed the beta-galactosidase reporter gene, with no observable toxicity. DNA concentrations >2 microgram/ml were essential for efficient gene delivery. This synthetic peptide provides a safe, readily standardised and flexible DNA vector system well suited to ex vivo gene delivery to neurons for experimental and clinical applications.
Collins
Louise
L
Department of Clinical Sciences, Institute of Liver Studies, Guy's, King's and St Thomas' School of Medicine, King's College Hospital, London, UK.
Asuni
Ayodeji A
AA
Anderton
Brian H
BH
Fabre
John W
JW
D000818:Animals; D018501:Antirheumatic Agents / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics*; D002411:Cation Exchange Resins / Q000494:pharmacology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D002738:Chloroquine / Q000494:pharmacology; D003435:Crotalid Venoms / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005260:Female; D005333:Fetus; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter / Q000187:drug effects / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics; D006026:Glycoside Hydrolases; D006388:Hemagglutinins; D006389:Hemagglutinins, Viral; D007202:Indicators and Reagents / Q000494:pharmacology; D008055:Lipids / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D009474:Neurons / Q000502:physiology*; D009842:Oligopeptides / Q000235:genetics* / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection / Q000379:methods*; D014764:Viral Proteins; D001616:beta-Galactosidase / Q000096:biosynthesis
D018501:Antirheumatic Agents; D002352:Carrier Proteins; D002411:Cation Exchange Resins; D003435:Crotalid Venoms; D006388:Hemagglutinins; D006389:Hemagglutinins, Viral; D007202:Indicators and Reagents; D008055:Lipids; C086724:Lipofectamine; D009842:Oligopeptides; D010446:Peptide Fragments; D014764:Viral Proteins; C458881:hemagglutinin fusogenic peptide, influenza virus; C458882:polylysine-molossin peptide; D002738:Chloroquine; D008156:Luciferases; D006026:Glycoside Hydrolases; C089138:beta-galactanase; D001616:beta-Galactosidase