FIGURE 1.
Gene targeting of 5′cSμ region into endogenous IgH V region locus. (A) Targeting strategy of 5′cSμ region. Restriction endonuclease map of the endogenous Igh locus is shown. The closed circle represents the Igh intronic enhancer (iEμ), and the closed boxes represent DQ52 and JH1–4 elements. Targeting construct was used to introduce the modified 5′cSμ allele (V-5′cSμ) into wt 129 ES cells. The XhoI–EcoRI fragment containing the DQ52 and JH1–4 elements was replaced by the V-5′cSμ cassette and the floxed neomycin gene. Closed triangles represent the loxP sites. Arrow indicates the VH186.2 promoter. An asterisk indicates the stop codon in the leader sequence. Striped box indicates 5′cSμ region. (B) Southern blot analysis of targeted ES cells. Left panel, EcoRI-digested ES cell DNA was hybridized with 3′ probe (JH probe). Right panel, HindIII-digested DNA before and after deletion of the neomycin gene was hybridized with 5′ probe A (DQ52 probe). Germline (GL) and targeted bands (in kb) are indicated by arrows. (C) Schematic of VB1–8 productive and V-cSμ passenger alleles. Top panel: Configuration of VB1–8 allele. The pattern-filled box indicates the VB1–8 exon sequence. The open box (L) indicates the leader of the VB1–8 exon. The V pro indicates the VH186.2 promoter. The oval box indicates Eμ. Bottom panel, Configuration of V-5cSμ allele. A 760-bp of the cSμ region replaced a large portion of the VB1–8 exon sequence with 21 bp of the V region exon and 19-bp JH2 exon left flanking the 5′cSμ region, and a stop codon (*) was introduced into the leader sequence (L). (D) Semiquantitative RT-PCR analysis of V-Cμ transcripts in unstimulated or stimulated B cells from VB1–8 or V-cSμ KI mice. The cDNA samples were prepared from unstimulated or stimulated B cells as described in Materials and Methods, and diluted in 1:5 serials for actin (1:5, 1:25, and 1:125) or 1:3 serials for V-Cμ transcripts (no dilution, 1:3 and 1:9). Representative data are shown from three independent experiments.