Programmed Cell Death

Nachbur, Ueli, Brumatti, Gabriela, Ueli Nachbur, Gabriela Brumatti

Ectopic expression of proteins involved in cell death pathways is an important tool to analyze their role during apoptosis or other forms of cell death. Lentiviral vectors offer the advantages of high rate of transduction and stable integration of donor DNA into the genome of the host cell, leading to reproducible and relevant readouts compared to classical overexpression by transfection of naked plasmid DNA.Here, we describe the production and application of lentiviral vectors to express cell death proteins in eukaryotic cells. A packaging cell line, usually HEK293T cells, is transfected with viral packaging plasmids and your gene of interest, which is flanked by long terminal repeat sites with an internal ribosome entry site in the 5'UTR (Un translated region). Virions are harvested from the supernatant and can be directly used to transduce target cells. Varied selection markers as well as a variety of promoters that regulate expression of the gene of interest make this system attractive for a wide range of application in many cell lines or in whole organisms.