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Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death
Published on: October 21, 2012
Use of gene knockouts in cultured cells to study apoptosis
1Department of Tumor Cell Biology, St. Jude Children's Research Hospital, 332 North Lauderdale, Memphis, Tennessee 38105, USA. jill.lahti@stjude.org
Abstract:
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.
Insights
The avian DT40 cell system efficiently creates gene knockouts in vertebrate cells for studying apoptosis. This powerful tool aids in understanding cell death pathways and can be adapted for human gene research.
Area of Science:
- Cell Biology
- Molecular Genetics
- Apoptosis Research
Background:
- The avian DT40 cell line is a unique model for genetic manipulation.
- High-frequency homologous recombination enables efficient gene "knockout" creation.
- Understanding gene function in apoptotic processes is crucial.
Purpose of the Study:
- To introduce a novel method for generating loss-of-function mutations in vertebrate cells.
- To utilize the DT40 system for dissecting apoptotic pathways and gene function.
- To explore the adaptability of the DT40 system for human gene studies.
Main Methods:
- Employing homologous recombination in DT40 chicken B lymphoma cells to knock out target genes.
- Utilizing tetracycline-responsive promoters for inducible cDNA expression and gene rescue.
- Transfecting knockout cells with cDNA libraries or expression constructs for complementation assays.
- Adapting the system for gene disruption in DT40/human hybrid cell lines.
Main Results:
- Demonstrated the efficacy of the DT40 system in generating loss-of-function mutations.
- Enabled the study of gene modulation in apoptosis and the ordering of cell death pathway components.
- Showcased the system's flexibility for functional domain delineation and complementation studies.
- Extended the system's applicability to human gene research.
Conclusions:
- The avian DT40 cell system is a versatile and powerful tool for genetic research, particularly in apoptosis.
- It facilitates the functional characterization of genes involved in cell death.
- The system's adaptability holds significant promise for advancing human gene function studies.
