Valentina C. Ciccarone

Improving the transfection efficiency of post-mitotic neurons

The transfection of post-mitotic cells, including primary cortical and hippocampal neurons, has proven for the most part to be inefficient. Methods such as DNA/Ca++ phosphate co-precipitation, electroporation, cationic lipids and micro-injection are often toxic to the cell and rarely give a transfection efficiency exceeding 3% of the surviving culture. Virus transfection methods using modified viruses such as adeno and semliki-forest virus have been shown to be more efficient but the procedures are often time consuming and virus infections may interfere with protein processing. In this study, we evaluated the transfection efficiency of cells from E18 rat embryonic cortical and hippocampal tissues using three cationic lipids: LIPOFECTAMINE, LIPOFECTAMINE Plus, and LIPOFECTAMINE 2000. The method of transfection was by a traditional reporter gene beta-galactosidase (pCMV.SPORT-beta-gal). Results show that out of the three cationic lipids tested, LIPOFECTAMINE 2000 allows for a significantly higher transfection efficiency. Transfection efficiency with LIPOFECTAMINE or LIPOFECT-AMINE Plus was <3%. In contrast, transfection efficiency with LIPOFECT-AMINE 2000 was approximately 20-25% with the cortical neurons and 25-30% with the hippocampal neurons.

Transfection of cultured eukaryotic cells using cationic lipid reagents.

The development of high‐efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipids has accelerated biological research in the studies of gene expression, control of cell growth, and cell lineage. Transfection mediated by cationic lipids is commonly used in industrial protein production as well as in some clinical gene therapy protocols. This unit describes how to perform transfection of adherent and suspension cells, insect cells, and RNA transfection using cationic lipid reagents. Curr. Protoc. Mol. Biol. 81:9.4.1‐9.4.17.

UNIT 9.4 Transfection of Cultured Eukaryotic Cells Using Cationic Lipid Reagents

The development of high‐efficiency methods for the introduction of functional genetic material into eukaryotic cells using cationic lipids has accelerated biological research in the studies of gene expression, control of cell growth, and cell lineage. Transfection mediated by cationic lipids is commonly used in industrial protein production as well as in some clinical gene therapy protocols. Replacing our previous unit on this topic, this new version describes how to perform transfection of adherent and suspension cells, insect cells, and RNA transfection using the cationic lipid system.