Abstract
A chicken DT40 B-cell line provides an excellent model system for the analysis of the function of genes related to B-cell antigen receptor (BCR) signaling. Crosslinking of BCR by ยต-chain-specific antibody stimulates DT40 cells to undergo apoptosis. Activation of protein-tyrosine kinases (PTKs) and intracellular calcium signaling are essential for BCR-induced apoptosis. Because DT40 B cells are highly proliferative and readily integrate exogenous DNA by homologous recombination, gene targeting is conveniently employed to inactivate genes of interest. This chapter describes procedures for (1) generation of knockout DT40 cell line, (2) analysis of surface BCR expression, (3) detection of tyrosine-phosphorylated proteins, (4) detection of intracellular calcium mobilization, and (5) reconstitution of depleted gene by transfection.
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References
Yasuda, T., Maeda, A., Kurosaki, M., et al. (2000) Cbl suppresses B cell receptormediated phospholipase C (PLC)-ฮณ2 activation by regulating B cell linker protein-PLC-ฮณ2 binding. J. Exp. Med. 191, 641โ650
Takata, M., Sabe, H., Hata, A., et al. (1994) Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways. EMBO J. 13, 1341โ1349.
Reth, M. and Wienands, J. (1997) Initiation and processing of signals from the B cell antigen receptor. Annu. Rev. Immunol. 15, 453โ479.
DeFranco, A. L. (1997) The complexity of signaling pathways activated by the BCR. Curr. Opin. Immunol. 9, 296โ308.
Tamir, I. and Cambier, J. C. (1998) Antigen receptor signaling: integration of protein tyrosine kinase functions. Oncogene. 17, 1353โ1364.
Kurosaki, T. (1999) Genetic analysis of B cell antigen receptor signaling. Annu. Rev. Immunol. 17, 555โ592.
Winding, P. and Berchtold, M. W. (2001) The chicken B cell line DT40: a novel tool for gene disruption experiments. J. Immunol. Methods 249, 1โ16.
Abdrakhmanov, I., Lodygin, D., Geroth, P., et al. (2000) A large database of chicken bursal ESTs as a resource for the analysis of vertebrate gene function. Genome Res. 10, 2062โ2069.
Fukagawa, T., Hayward, N., Yang, J., et al. (1999) The chicken HPRT gene: a counter selectable marker for the DT40 cell line. Nucleic Acids Res. 27, 1966โ1969.
Wang, J., Takagaki, Y., and Manley, J. L. (1996) Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability. Genes Dev. 10, 2588โ2599.
Fukagawa, T. and Brown, W. R. (1997) Efficient conditional mutation of the vertebrate CENP-C gene. Hum. Mol. Genet. 6, 2301โ2308.
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Yasuda, T., Yamamoto, T. (2004). Analysis of B-Cell Signaling Using DT40 B-Cell Line. In: Gu, H., Rajewsky, K. (eds) B Cell Protocols. Methods in Molecular Biology, vol 271. Humana Press. https://doi.org/10.1385/1-59259-796-3:261
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DOI: https://doi.org/10.1385/1-59259-796-3:261
Publisher Name: Humana Press
Print ISBN: 978-1-58829-347-3
Online ISBN: 978-1-59259-796-3
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