Studies on the regulatory mechanisms of V (D) J rearrangement using gene-inserted mice.

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In order to understand the regulatory mechanisms of immunogoloulin heavy chain rearrangement, studies have been performed using a mutant mouse strain which carries a functionally rearranged V_H gene (V_HT15) inserted in the V_H locus on one chromosome (T15i/+ mice). I have shown followings. 1. In T15i/+ mice, B cells bearing the V_HT15 product occupies around 40% in the peripheral B cell compartment. In contrast, V_HT15^+B cells comply of around 80% of the B cell pool in T15i/+ mice carrying additional mutation in the IL-7 receptor loci (T15i/+IL-7R^<-/->), indicating that IL-7 is required for the survival of pro-B cells which undergo V(D)J rearrangement, so that in the absence of IL-7, pro-B cells cannot survive long enough to perform secondary rearrangements to disrupt the inserted V_H gene and generate functional V_H gene on the wild-type chromosome. 2. A kappa-chain transgene was additionally introduced into the T15i/+IL-7R^<-/-> mice. Even in these mice, the percentage of VHT15+B cells are still as high as that in non-transgenic T15i/+IL-7R^<-/-> mice. Moreover, the reduction of the B cell number due to the lack of IL-7 signals could not be restored by introducing both heavy and light cahins. These results indicate that the IL-7 signals function independently of surface Ig signals. 3. Secondary rearrangements on the mutant chromosome which destroy the inserted VH gene in T15i/+ mice might be due to the presence of the neomycin resistant cassette (neo) present upstream of the inserted VH gene which had been used as a selection marker in gene targeting. However, deletion of the ned gene using FLIP-FRT system did not alter the frequency of such secondary rearrangement. Thus, the secondary rearrangements occur as a result of the unique structure of VHT15 gene itself (for example, the sequence or the transcriptional activity of the accompanying promotor).