Liposome-Mediated Transfection

INTRODUCTION The efficient delivery of exogenous DNA to cells for expression and function studies is an essential technique of modern cell biology. Cationic liposomes are the most widely used class of carrier for transfection and a number of products are available commercially: Lipofectin (a 1:1 mixture of DOTMA [ N -[1(2,3-dioleoyloxy)propyl]- N , N , N -trimethylammonium chloride] and DOPE [dioleoyl phosphatidylethanolamine]), Transfectam, and DOTAP [1,2-dioleoyloxy-3-trimethylammonium propane]:DOPE. However, all these reagents are costly; it can be less expensive to purchase purified lipids and formulate homemade transfection reagents by adding equimolar amounts of each lipid suspended in chloroform, mixing, and drying under inert gas. The reagents can be stored until they are reconstituted in an appropriate buffer. Although each manufacturer provides detailed instructions for the use of its reagent, the following general steps are used for essentially all formulations; times, concentrations, and volumes might differ slightly. The following volumes are sufficient for transfecting a 35-mm dish or one well of a six-well dish.