PHOSPHATE-ENHANCED TRANSFECTION OF CATIONIC LIPID-COMPLEXED MESSENGER-RNA AND PLASMID DNA

Cationic lipid-mediated gene transfer has been shown to be a competent albeit inefficient mechanism of promoting cellular gene transfer. One way to improve the efficacy of cationic lipid-mediated transgene expr ession is to optimize conditions for complex formation between the lip ids and nucleic acids. In this report we describe the beneficial effec ts of using phosphate buffer to precondition lipofectin (a 1:1 (w/w) m ixture of [1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA), and dioleoyl phosphatidylethanolamine (DOPE)) prior to comple xing with plasmid DNA or mRNA. Under such optimized conditions we stud ied the kinetics of DNA- and RNA-mediated transgene expression in a hu man osteosarcoma cell line (HOS), Preincubation of lipofectin in phosp hate buffer resulted in up to 26- and 56-fold increases in luciferase expression from plasmid DNA and mRNA, respectively. Addition of chloro quine (50 mu M), which enhanced plasmid-mediated gene delivery 3-fold, was synergistic with phosphate resulting in an additional 46-fold inc rease in luciferase expression. The preincubation with phosphate short ened both the time required for cellular uptake and the time to achiev e maximal transgene expression. Optimal transfection was achieved in t he presence of 30-50 mM phosphate, at pH 5.6-6.8 under which the phosp hate anion is divalent. The effect of phosphate anion was specific in that monovalent Cl- and acetate anions were not stimulatory. These res ults demonstrate that divalent phosphate anion plays a stimulatory rol e during complex formation and transfection when cationic lipids come in contact with negatively charged nucleic acids and cell membranes. T hese findings delineate specific conditions which dramatically enhance transfection efficiency for both DNA and mRNA, and provide an effecti ve procedure for gene transfection studies. (C) 1998 Elsevier Science B.V.