Optimization of mouse embryonic stem cell transfection for the new mutagenesis methods

Abstract

Embryonic stem (ES) cells are derived from the inner cell mass of blastocysts, an early-stage of pre-implantation embryo, and are capable of unlimited, undifferentiated proliferation in vitro. They are pluripotent, meaning they are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. ES cells are key tools for genetic engineering, development of stem cell-based therapies and basic research on pluripotency and early lineage commitment. The use of stem cells as therapeutics to treat genetic defects depends on how efficient are the approaches to manipulate their genome. Traditional non-viral strategies are generally less efficient in delivering DNA and initiating gene expression, but they are safer, cheaper, producible easily in large quantities and have higher genetic material carrying capacity. Therefore, FuGENE® HD (Promega), SuperFect® (Qiagen), Lipofectamine® 2000 (Invitrogen) and also electroporation were used to transiently transfect fluorescently labelled expression vectors into an mES cell line in order to optimize a reliable and efficient protocol that could be applied for some of the new mutagenesis methods. In most of the new site-directed mutagenesis techniques, more than one plasmid has to be introduced into the cell. For this reason, co-transfection and single transfection efficiencies of plasmids encoding the mCherry fluorescent protein and the EGFP were simultaneously determined. Transfection and co-transfection efficiencies of 52-83% were found for FuGENE® HD transfection reagent, which was shown to be most efficient and reliable. In addition, in 90-93% of the co-transfected colonies both fluorescent proteins were co-expressed. This optimized transfection protocol was followed to successfully assess a new recombinase mediated cassette exchange (RMCE) system in an mES RMCE-in cell line as a more attractive alternative to the commercial Flp-in cell lines