Ig V region hypermutation in B cell hybrids mimics in vivo mutation and allows for isolation of clonal variants.

In order to investigate the regulation of Ig hypermutation, we have established a cell culture system in which reversion of a V region stop codon in a stably transfected Ig gene permits the quantitation of mutation rates by fluctuation analysis. Transfected heavy chain V regions associated with the mu constant region undergo low rates of mutation in the NSO plasmacytoma cell line and a moderate rate of mutation in the 18.81 pre-B cell line. Most of the hybrids created by fusing these two cell lines resembled the non-permissive NSO cell line, though a few hybrids had constitutive V region mutation rates that were even higher than 18.81 and similar to the high rates of mutation that occur in vivo (Green, N. S., Rabinowitz, J. L., Zhu, M., Kobrin, B. J. and Scharff, M. D. (1995) Proc. Nat. Acad. Sci. (USA) 92, 6304 6308). Characterization of these hybrids now demonstrates that the transfected genes were integrated outside of the Ig locus. Mutation was due to multiple single base pair replacements in the V region and not the C region, was ongoing and often arose in hot spot motifs described by V region hypermutation in vivo. Subcloning of unstable hybrids allowed for the isolation of highly related clones with 44-70-fold different mutation rates. These results suggest that V region hypermutation in this mode in vitro systems is under both positive and negative regulation.