A REPORTER GENE TO ANALYZE THE HYPERMUTATION OF IMMUNOGLOBULIN GENES

The affinity maturation of antibodies is driven by somatic hypermutati on which is localized to specific segments of the coding genes. The in formation available on this process derives from studies in vivo. With the intention of developing new approaches, we have constructed a fus ion gene between a kappa chain and a selectable neomycin resistance ge ne, neo(r). The neo(r) gene, which includes the SV40 small t intron an d polyadenylation site, but not the upstream elements nor its first 12 amino acids, is an in-frame substitution of the FR2-CDR3 fragment of a rearranged V kappa Ox1-J kappa 5 gene. Expression of neo(r) activity is therefore dependent on the upstream immunoglobulin sequence. A sto p codon was placed in the CDR1 region so that only mutants survive tre atment with geneticin sulphate (G418). The effectiveness of the system was tested by transfecting the NS0 myeloma cell line and isolating sp ontaneous mutants. Neomycin-resistant clones arose at an estimated rat e of 1 x 10(-8)/cell division, and over 90% were authentic structural mutants. Unlike the somatic hypermutations, the majority arose by in-f rame deletions including the stop codon, although up to 30% involved a point mutation. The reporter gene was then modified by substituting a ll the sequences downstream of the J kappa 5 with others known to be r equired for full hypermutation in vivo. Different cell lines were tran sfected and G418-resistant clones analyzed. No significant increase in the rate of reversion or in the generation of point mutations versus deletions was detected, even using conditioned culture medium. In the presence of azacytidine however, a mutant involving multiple events (s ingle base addition and deletion plus two point mutations) was detecte d. The reporter gene system therefore seems suitable to test culture c onditions and modifications of the host cells aimed at the derivation of an in vitro assay of somatic hypermutation.