Use of gene knockouts in cultured cells to study apoptosis

The avian DT40 cell system represents a novel method to generate loss of fu nction mutations in vertebrate cells. These chicken B lymphoma cells underg o homologous recombination at very high frequencies and can thus be used to "knock out" genes believed to function in apoptotic processes. The knockou t cells can then be used to determine how the cell death process is modulat ed after induction of apoptosis and to order components in cell death pathw ays. The system can be further modified, using tetracycline-responsive prom oters, to allow expression of wild-type cDNAs to rescue "knockout cells" if the gene of interest is essential. Alternatively, cDNA expression construc ts containing mutations or deletions in the cDNA encoding the absent protei n can be used to delineate functional domains. cDNA expression libraries or known proteins believed to function downstream of the target in a signal t ransduction pathway could also be transfected into the knockout cell line, and the resultant cells could be assayed for complementation and/or rescue of the apoptotic alteration/ defect. Finally, the system has recently been adapted to allow disruption of human genes in DT40/human hybrid cell lines thereby potentially extending this system for use in studying human genes. (C) 1999 Academic Press.