Fig. 4.

V_HQ52^NT is edited through VH replacement. (A) CDR3 nucleotide sequence of V_HQ52^NT. Cryptic heptamer and n-nucleotides are indicated, respectively, in red and gray. (B) PCR strategy to identify VH replacements. Arrows indicate PCR primers. Colored blocks symbolize V_H families. An oligonucleotide annealing to a unique sequence (FP) within the CDR3 of V_HQ52^NT was used as a reverse primer. (C) Pie representation of V_H gene use in VH replacements cloned from splenic IgM⁺ B cells of V_HQ52^NT-HT mice (n = 4). Numbers indicate frequency of rearrangements carrying the indicated V_H genes. PCR primers used for the analysis are indicated. (D) CDR3 nucleotide sequence of representative VH replacements cloned from IgM⁺ B cells of V_HQ52^NT-HT mice using the V_HX24.a1.84 donor V_H gene. First line shows CDR3 sequence of the original V_HQ52^NT rearrangement. Underlined are V_HQ52^NT FP sequences indicated, respectively, as FP1 and FP2. De novo added n-nucleotides are labeled in brown. cRSSs are indicated in red. (E) Frequency of unique VH replacements cloned from IgM⁺ splenic B cells of V_HQ52^NT-HT mice, as assessed through CDR3 sequence analysis. Replacements using the same V_H germ-line gene were grouped. (F) Southern blotting quantification of V_HQ52^NT gene copy number in splenic IgA⁺ B cells of V_HQ52^NT homozygous (HO) mice. Data were normalized for DNA input and represented as relative to V_HQ52^NT copy number in liver cells of transnuclear mice. (G) Representative flow cytometric analysis of splenic CD19⁺ B cells of V_HQ52^NT;B1-8f double IgH insertion mice (n = 4). Number within dot plot refers to frequency of IgM-only B cells.