Position-dependent inhibition of class-switch recombination by PGK-neor 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 C mu, C delta, C gamma 3, C gamma 1, C gamma 2b, C gamma 2a, C epsilon, and C alpha. Expression of C-H genes downstream of C delta involves class-switch recombination (CSR), a process that is targeted by germ-line transcription (GT) of the corresponding Ctl gene. Previously, we demonstrated that insertion of a PGK-neo(r) cassette at two sites downstream of Ca inhibits, in cultured B cells, GT of and CSR to a subset of Cw genes (including C gamma 3, C gamma 2a, C gamma 2b, and CE) 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 I gamma 2b locus inhibits GT of and CSR to the upstream C gamma 3 gene, but has no major effect on the downstream C gamma 2a and CE genes. Moreover, replacement of the C epsilon c persons with a PGK-neo(r) cassette in the opposite transcriptional orientation also inhibits, in culture, GT of and CSR to the upstream C gamma 3, C2b, and Cy2 alpha genes..is with the PGK-neo(r) insertions 31 of Car studied previously, the Cy1 and Ca genes were less affected by these mutations both in culture and in mice, whereas the C gamma 2b gene appeared less affected bl vivo. Our findings support the existence of a long-range 3' IgH regulatory region required for GT of and CSR to multiple CH genes and suggest that PGK-neo(r) cassette insertion into the locus short circuits the ability of this region to facilitate GT of dependent CH genes upstream of the insertion.