Immunoglobulin gene conversion and hypermutation assay by FACs.

Abstract

Immunoglobulin (Ig) gene conversion and hypermutation rates can be quantified by FACS. The spontaneous DT40 variant, C118, and C118-derived DT40 mutants can be used to analyze gene conversion phenotype. The C118 has a frameshift in its rearranged V segment. When this frameshift mutation is repaired by pseudogene-templated gene conversion, sIgM(-) DT40 reverts to sIgM (+) (Ig reversion assay). The sIgM (+) pseudogene knockout DT40 clone can be used to analyze Ig hypermutation phenotype. Pseudogene knockout DT40 accumulates Ig hypermutation, rapidly generating sIgM (-) population (sIg loss assay). These changes of sIgM expression can be measured by FACS. To minimize the fluctuation of gene conversion or hypermutation events, it is needed to analyze many subclones of the DT40 mutant for calculating average sIgM (-) or (+) percentage.