SOMATIC HYPERMUTATION OF AN ARTIFICIAL TEST SUBSTRATE WITHIN AN IG-KAPPA TRANSGENE

We have characterized a novel substrate for somatic hypermutation, con firming that non-Ig sequences can be targeted for mutation and demonst rating that this substrate allows for the rapid assay for mutations, A n artificial sequence containing alternating EcoRV and PvuII sites (EP S) was inserted into the V kappa 167 transgene, which is known to be a target for mutation. To assay for somatic hypermutation, the EPS is a mplified using flanking transgene primers, and the PCR product is subs equently digested with either EcoRV or PvuII. A mutation is seen as th e appearance of a larger fragment, indicating a base change in a restr iction enzyme site, The original transgene, V kappa 167/EPS, showed ev idence of a low level of mutation in both splenic hybridomas and Peyer 's patch-derived or immunized splenic B220(+) cells with high peanut a gglutinin levels, Two derivative lines of V kappa 167/EPS were made, V kappa 167/POX and V kappa 167/PEPS. While none of the V kappa 167/POX transgenic lines demonstrated mutation, the V kappa 167/PEPS transgen e was highly mutated in B220(+) splenic B cells with high peanut agglu tinin levels at a frequency similar to that of endogenous Ig genes, An analysis of splenic RNA from the unimmunized transgenic mice indicate d that the levels of stable message in splenic B cells could not be co rrelated with the mutation seen in GC B cells. The mutable V kappa 167 /PEPS transgenic line is a unique? tool to study somatic hypermutation in vivo.