Position-dependent inhibition of class-switch recombination by PGK-neo^r cassettes inserted into the immunoglobulin heavy chain constant region locus

The Ig heavy chain (IgH) constant region (C_H) genes are organized from 5′ to 3′ in the order Cμ, Cδ, Cγ3, Cγ1, Cγ2b, Cγ2a, Cε, and Cα. Expression of C_H genes downstream of Cδ involves class-switch recombination (CSR), a process that is targeted by germ-line transcription (GT) of the corresponding C_H gene. Previously, we demonstrated that insertion of a PGK-neo^r cassette at two sites downstream of Cα inhibits, in cultured B cells, GT of and CSR to a subset of C_H genes (including Cγ3, Cγ2a, Cγ2b, and Cε) that lie as far as 120 kb upstream. Here we show that insertion of the PGK-neo^r cassette in place of sequences in the Iγ2b locus inhibits GT of and CSR to the upstream Cγ3 gene, but has no major effect on the downstream Cγ2a and Cε genes. Moreover, replacement of the Cε exons with a PGK-neo^r cassette in the opposite transcriptional orientation also inhibits, in culture, GT of and CSR to the upstream Cγ3, Cγ2b, and Cγ2a genes. As with the PGK-neo^r insertions 3′ of Cα studied previously, the Cγ1 and Cα genes were less affected by these mutations both in culture and in mice, whereas the Cγ2b gene appeared less affected in vivo. Our findings support the existence of a long-range 3′ IgH regulatory region required for GT of and CSR to multiple C_H genes and suggest that PGK-neo^r cassette insertion into the locus short circuits the ability of this region to facilitate GT of dependent C_H genes upstream of the insertion.