Use of gene knockouts in cultured cells to study apoptosis.
The avian DT40 cell system represents a novel method to generate loss of function mutations in vertebrate cells. These chicken B lymphoma cells undergo homologous recombination at very high frequencies and can thus be used to "knock out" genes believed to function in apoptotic processes. The knockout cells can then be used to determine how the cell death process is modulated after induction of apoptosis and to order components in cell death pathways. The system can be further modified, using tetracycline-responsive promoters, to allow expression of wild-type cDNAs to rescue "knockout cells" if the gene of interest is essential. Alternatively, cDNA expression constructs containing mutations or deletions in the cDNA encoding the absent protein can be used to delineate functional domains. cDNA expression libraries or known proteins believed to function downstream of the target in a signal transduction 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.
Lahti
J M
JM
Department of Tumor Cell Biology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, Tennessee 38105, USA. jill.lahti@stjude.org
D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D015139:Blotting, Southern; D002645:Chickens; D019938:Cyclin D1 / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D015723:Gene Library; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D006822:Hybrid Cells; D009154:Mutation; D011995:Recombination, Genetic; D015398:Signal Transduction / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D019938:Cyclin D1; D003907:Dexamethasone