Analysis of B-Cell Signaling Using DT40 B-Cell Line

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 �-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.