Since

Ig membrane expression on B lymphocytes is required for cell survival 11, 12, targeting IgM exons or the JH locus with ZFN was expected to generate non-homologous end joining mutations resulting in Ig-deficient rats and thus lacking TSA HDAC cell line mature B cells. In this manuscript, we describe the phenotype of rats homozygous for a truncation in Cμ1 and, separately, deletion of the JH locus. Both lines show no detectable Ig production and mature B-cell development. The availability of B-cell-deficient rats will permit to gain new insights of Ig function and development in health and disease. In addition, ZFN technology paves the way for simpler gene replacement and transgenic studies with the immediate aim of expressing human Ab repertoires in the rat. Among several rat lines with IgM CH1 domain mutations 8,

rat line 19 was breed to homozygocity. The mutation in this rat line comprised a 64 bp deletion in both alleles of the IgM CH1 domain gene see more (Fig. 1A, left) and no additional mutations in any of the ten genomic sequences most homologous to the one targeted 8. Analysis of IgM mRNA by RT-PCR of JH1-Cμ transcripts showed a shorter transcript in rats homozygous for IgM mutation (IgM KO rats) compared with WT (Fig. 1B, left). Analysis of IgG transcripts using RT-PCR of JH-Cγ showed the absence of mRNA in IgM KO rats and a strong signal of the expected size in WT rats (Fig. 1B, left). Heterozygous IgM KO rats showed the presence of IgM and IgG transcripts (data not shown). Digestion of the JH-Cμ amplicon with DdeI resulted in the generation of a smaller band due to the 64 bp deletion (Fig. 1B, right). Sequencing of JH-Cμ mRNA isolated from IgM KO rats showed a deletion of 64 bp and the generation of a stop codon (Fig. 1C). Microinjection of rat zygotes with ZFN mRNA specific for the JH locus resulted in the generation of a mutant animal with a 2465 bp DNA deletion, spanning Phloretin the entire locus (Supporting Information Data 1). In homozygous JH locus, mutant rats’ analysis of mRNA using primers spanning several VH or JH sequences to μCH2

(Fig. 1D) or Cγ sequences (data not shown) did not reveal detectable levels of transcripts. These results indicate that IgM KO rats have a deletion in the Cμ1 domain that generated a stop codon, resulting in shorter IgM transcripts and no IgG transcripts. Rats homozygous for J deletion (JH KO rats) showed a large deletion and no detectable IgM or IgG transcripts. ELISA revealed undetectable levels for all Ig isotypes in IgM or JH KO rats analyzed (Fig. 2A). Heterozygous IgM KO animals and WT rats showed normal levels of IgM (1 246±81 μg/mL), IgG (6 060±1 356 μg/mL), IgA (65±5 μg/mL) and IgE (2 845±1 110 ng/mL). In mice, mutations in the IgM Cμ1 exon have resulted in alternative splicing of the mutated region and shorter μ-chains were produced 13.