STABLE TRANSFECTION OF MAMMALIAN-CELLS BY SYRINGE-MEDIATED MECHANICALLOADING OF DNA

We show that mammalian cells can be stably transfected by a mechanical loading procedure in which cells are forced through a small opening i n the presence of DNA. A suspension of cells and plasmid DNA in growth medium was passed up and down through a 30-gauge needle attached to a 1-ml syringe. Cells were immediately plated at appropriate densities for subsequent selection for stable expression of a marker gene. Two r odent cell Lines, Chinese hamster ovary and mouse Ltk(-) cells, were s uccessfully transfected with an efficiency of about one transfectant p er 5 x 10(4) cells. The human HeLa cell line was transfected with a so mewhat lower efficiency. Pluronic F-68, a detergent believed to aid in healing of membrane injuries, had no beneficial effect when present d uring the loading procedure. Successful transfection was accomplished using three different genes as selectable markers. Southern blotting a nalysis revealed that transfectants contained one or very few copies o f the introduced DNA construct integrated into the genome. Several tra nsfectants were demonstrated to remain stable for more than 20 generat ions of growth in the absence of selection. This procedure is fast, ec onomical, and of general utility. (C) 1998 Academic Press.