21]Extrachromosomal substrates to study immunoglobulin heavy chain class switch recombination in mammalian B cells

Abstract

Publisher Summary This chapter discusses the use of extrachromosomal substrates to study immunoglobulin heavy chain class switch recombination in mammalian B cells. Immunoglobulin heavy chain class switch recombination is a regulated recombination event that joins an expressed immunoglobulin heavy chain variable (VDJ) region to a new downstream constant (C) region of a different class or isotype. The result of switch recombination is to modify the antigen clearance properties of an immunoglobulin molecule without affecting its specificity for antigen. The chapter presents an assay for genetic elements and factors that regulate switch recombination. This assay measures the recombination of extrachromosomal substrates that carry sequences from the S μ and S γ 3 switch regions flanking a conditionally lethal marker, the leftward (P L ) promoter of bacteriophage λ. Constructs carrying λP L can be propagated in lysogenic strains of Escherichia coli , where the endogenous c I repressor shuts off transcription from APL, but they cannot give rise to ampicillin-resistant colonies in nonlysogens. The switch substrate recombination assay is rapid and the recombination substrates can be readily manipulated to test the effect of different sequences and regulatory elements on recombination. The assay also allows easy recovery of recombination products for analysis. For these reasons, it has provided a valuable tool in the study of cis -acting elements that regulate switch recombination.