Analysis of Modulation of Immunoglobulin Gene Expression

Abstract

Immunoglobulins (Ig) are critical in maintaining host immunity to a variety of pathogens. Regulation of Ig expression is a complex process involving transcriptional regulation of different Ig gene loci by many transcription factors and transcriptional regulatory regions. This complexity suggests many possible molecular targets for immunotoxicants. Therefore, thorough evaluation of chemical-induced modulation of Ig expression may necessitate multiple experimental approaches evaluating: (1) number of B cells secreting antibodies by antibody-forming cell response or plaque assay; (2) concentration of total secreted antibodies by enzyme-linked immunosorbent assay (ELISA); (3) cellular proliferation and viability by cell count measurements, [(3)H] thymidine incorporation, and trypan blue exclusion; (4) Ig mRNA expression by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR); (5) transcriptional activity of specific Ig regulatory regions by reporter gene analysis; and (6) transcription factor binding to specific Ig regulatory regions by electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP). These experimental approaches are discussed in the unit, with detailed description of EMSA, EMSA-western analysis, and isolation of nuclear protein.