Stable transfection of mammalian cells by syringe-mediated mechanical loading 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 in 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 rodent cell lines, Chinese hamster ovary and mouse Ltk- cells, were successfully transfected with an efficiency of about one transfectant per 5 x 10(4) cells. The human HeLa cell line was transfected with a somewhat lower efficiency. Pluronic F-68, a detergent believed to aid in healing of membrane injuries, had no beneficial effect when present during the loading procedure. Successful transfection was accomplished using three different genes as selectable markers. Southern blotting analysis revealed that transfectants contained one or very few copies of the introduced DNA construct integrated into the genome. Several transfectants were demonstrated to remain stable for more than 20 generations of growth in the absence of selection. This procedure is fast, economical, and of general utility.