Transfection of epithelial cells is enhanced by combined treatment with mannitol and polyethyleneglycol.

Abstract

Gene transfer efficiency drops significantly when polarized mammary epithelial cells are transfected instead of actively growing cells. However, fully differentiated cells are the targets for gene transfer in many in vivo applications. Therefore, a simple and effective method for the transfection of polarized mammary epithelial cells in confluent monolayers was developed. Reporter gene plasmids were complexed with polyethylenimine with an average molecular weight of 25 kDa (PEI 25), or other agents, to transfect confluent monolayers of ovine mammary epithelial cells (OMEC II) or human carcinoma cells (CaCo-2) in vitro. The improved technique included pretreatment of the cells with a hyperosmotic mannitol solution (7%) which caused a loosening of the tight contacts between the cells. Alternatively, the mannitol shock could be replaced by a short treatment with trypsin or EDTA. In addition to the pretreatment, 12.5% polyethyleneglycol with an average molecular weight of 8000 kDa (PEG 8000) was included in the transfection mixture containing the DNA complexes. The combined application of mannitol and PEG resulted in a very reliable 5- to 30-fold increase in reporter gene expression in OMEC II and CaCo-2 cells, but not K562 cells (an example of another cell type). The improved technique can also be combined with other polymer-based transfection agents. The transfection rate was enhanced for confluent monolayer cells with fully developed epithelial polarity but also for subconfluent, growing epithelial cell cultures. A novel transfection protocol for epithelial cells is presented. The combined treatment of cells with mannitol and polyethyleneglycol results in substantial enhancement of in vitro transfection of epithelial cell lines.