An expressed neor cassette provides required functions of the Iγ2b exon for class switching.

Germline CH transcripts initiate from a promoter upstream of a non-coding I exon, proceed through the switch (S) region and terminate downstream of the associated CH exons. To elucidate the role of germline transcription in Ig heavy chain class switch recombination (CSR), we used gene targeting in embryonic stem (ES) cells to replace most of the Iγ2b exon from immediately 3′ of the majority of transcription initiation sites to beyond its donor splice site with a PGK-neor gene inserted in the same transcriptional orientation as the endogenous unit. The mutation was introduced into both alleles of ES cell lines (referred to as Iγ2bN/N) and the neor gene was deleted (referred to as Iγ2b-/-) by the IoxP/Cre method. These mutations were assayed for effects on CSR in B cells derived via RAG-2-deficient blastocyst complementation. Iγ2b-/- B cells lacked ability to switch to IgG2b both in vivo and in vitro, and, correspondingly, showed no germline transcription through the Iγ2b exon, Sγ2b region. In contrast, Iγ2bN/N B cells switched at normal levels IgG2b and showed substantial transcription through the Sγ2b and Cγ2b regions. Taken together, these results show that the Iγ2b sequences, per se, are not necessary for mediating CSR since a transcribed PGK-neor gene can replace its function. However, the deleted portion of the Iγ2b exon and splice donor site apparently contain sequences necessary for efficient germline gene transcription and thus for CSR to IgG2b. [ABSTRACT FROM PUBLISHER]