Abstract
The chicken B cell line DT40 has been widely used as a model system for reverse genetics studies in higher eukaryotes, because of its advantages including efficient gene targeting and ease of chromosome manipulation. Although the genetic approach using the RNA interference technique has become the standard method particularly in human cells, DT40 still remains a powerful tool to investigate the regulation and function of genes and proteins in a vertebrate system, because of feasibility of easy, rapid, and clear genetic experiments. The use of DT40 cells for DNA repair research has several advantages. In addition to canonical assays for DNA repair, such as measurement of the sensitivities toward DNA damage reagents, it is possible to measure homologous recombination and translesion synthesis activities using activation-induced deaminase (AID)-induced diversification of the immunoglobulin locus. In this chapter, we would describe a detailed protocol for gene disruption experiments in DT40 cells.
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Acknowledgements
The protocols resented here is the combinations of those developed through the work of our previous and current colleagues. The authors’ work was supported in part by Grants-in aid from the Ministry of Education, Science, Sports, and Culture of Japan (MI and MT). Financial support were also provided by the Uehara Memorial Foundation (MT), Takeda Foundation (MT), the Ichiro Kanehara Foundation (MI), and the Mochida Memorial Foundation for Medical and Pharmaceutical Research (MI).
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Ishiai, M., Uchida, E., Takata, M. (2012). Establishment of the DNA Repair-Defective Mutants in DT40 Cells. In: Bjergbæk, L. (eds) DNA Repair Protocols. Methods in Molecular Biology, vol 920. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-998-3_4
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DOI: https://doi.org/10.1007/978-1-61779-998-3_4
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