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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]

The IgH locus intronic enhancer (Eμ, located in the intron between the JH segments and the Cμ gene, and flanked by two matrix attachment regions (MAR), has been shown to be a major regulator of IgH gene transcription and VDJ recombination. To define the potential role of Eμ, plus MAR in class switch recombination (CSR), we generated IgG-expressing hybridomas from B cells heterozygous for mutations that delete all of these elements or replace them with a neor gene and analyzed the switch status of the mutated IgH loci. Eμ/MAR-deleted IgH loci displayed a highly significant, although not complete, decrease in CSR when compared to unmutated loci in normal hybridomas. Surprisingly, mutant loci with a pgk promoter-driven neor gene replacing the Eμ/MAR showed relatively normal switch frequency. These findings indicate that the Eμ/MAR region plays a significant, but not necessary role in facilitating class switching at the μ locus. Potential mechanisms for these findings are discussed. [ABSTRACT FROM PUBLISHER]

To investigate potential roles of the RAG-1 and RAG-2 gene products in Ig heavy chain class recombination (CSR), we have generated RAG-1-/- and RAG-2-/- mice which contain both a rearranged IgHC V(D)J gene (referred to as B1-8) inserted into the endogenous Ig heavy chain (HC) locus in place of the JH segments, and a rearranged δ1 light chain (LC) transgene (which are referred to as RAG-1-/-B1-8δ and RAG-2-/-B1-8δ mice respectively). The B1-8 HC gene and δ LC genes encode proteins that associated to form a complete Ig molecule, the expression of which leads to substantial reconstitution of the peripheral B cell compartments of RAG-1-/-B1-8δ and RAG-2-/-B1-8δ mice. Both RAG-1-/-B1-8δ and RAG-2-/-B1-8δ mice have relatively normal levels of the various IgG isotypes, but greatly reduced levels of serum IgM and IgA compared to normal littermates. Furthermore, RAG-1-/-B1-8δ and RAG-2-/-B1-8δ B cells activated in vitro with lipopolysaccharide (LPS) or LPS plus IL-4 responded similarly to control B cells with respect to surface expression and secretion of IgG3, IgG1, IgG2b, IgG2a and IgE, but again were deficient in the secretion of IgM. Together, these findings indicate that neither RAG-1 nor RAG-2 expression is required for efficient class switching to most HC isotypes in B cells. [ABSTRACT FROM PUBLISHER]