PCR-BASED ANALYSIS OF THE MURINE IMMUNOGLOBULIN HEAVY-CHAIN REPERTOIRE

The immunoglobulin heavy-chain repertoire has been mainly analysed by studying the proportion of genes belonging to each of the 14 described families, in terms of the expressed immunoglobulin molecules. Althoug h the proportion of each variable gene family is kept stable throughou t adult life and in different mice of the same strain, little informat ion is available on the clonal representation in the repertoire of act ivated B cells. We describe here a new method that permits an approach to this question by separating the products of a polymerase chain rea ction covering the V-H-D-J(H) junction of the immunoglobulin heavy-cha in gene in a sequencing gel, thereby allowing discrimination of differ ent rearrangements (according to their length) using a given J(H) and one of the member of a given V-H family. Using this method, we show th at it is possible to obtain a precise overview of the repertoire of ac tivated B cells, at the mRNA level, as well as the potential repertoir e, from a study of the DNA. We also show that this approach permits th e detection of an induced immune B cell response and studies of emergi ng dominant specific B cell clones.