Cyclic amphipathic peptide-DNA complexes mediate high-efficiency transfection of adherent mammalian cells.
A DNA transfection protocol has been developed that makes use of the cyclic cationic amphipathic peptide gramicidin S and dioleoyl phosphatidylethanolamine. The DNA complex is formed by mixing gramicidin S with DNA at a 1:1 charge ratio and then adding phosphatidylethanolamine at a lipid/peptide molar ratio of 5:1. The complex mediates rapid association of DNA with cells and leads to transient expression levels of beta-galactosidase ranging from 1 to 30% of the transfected cells, with long-term expression being about an order of magnitude lower. The respective roles of peptide and phospholipid are not yet resolved but optimal transfection requires both the cyclic peptide and the hexagonal phase-competent phospholipid PtdEtn. Transfection in CV-1 cells is not affected by lysomotrophic agents, which suggests that DNA entry into the cell is via the plasma membrane. This technique that is simple, economical, and reproducible mediates transfection levels up to 20-fold higher than cationic liposomes in adherent mammalian cells.
Legendre
J Y
JY
School of Pharmacy, University of California, San Francisco 94143-0446.
Szoka
F C
FC
D000818:Animals; D002412:Cations; D002448:Cell Adhesion; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006096:Gramicidin / Q000378:metabolism / Q000633:toxicity; D000882:Haplorhini; D006801:Humans; D008081:Liposomes; D008156:Luciferases / Q000235:genetics; D046911:Macromolecular Substances; D051379:Mice; D010456:Peptides, Cyclic / Q000378:metabolism* / Q000633:toxicity; D010743:Phospholipids / Q000378:metabolism; D014162:Transfection / Q000379:methods*
D002412:Cations; D008081:Liposomes; D046911:Macromolecular Substances; D010456:Peptides, Cyclic; D010743:Phospholipids; D006096:Gramicidin; D004247:DNA; D008156:Luciferases