Generation of a bicistronic VJCκ+VDJ_H CGG-specific knock-in mouse. (a) Schematic representation of the bicistronic VJCκ-2A-VDJ_H targeting cassette used to generate the Igh^CGG allele, before and after targeting to the Igh locus. The total insert size is 1.7 Kbp. The 3′ probe used for Southern blot is indicated. s.d., splice donor; E, EcoRV. Segments are not drawn to scale. (b) ELISA showing binding to IgY of recombinant WT anti–CGG mAb (rCGG) and recombinant anti–CGG mAb with the 2A cleavage product attached to the Cκ C terminus (rCGG 2A). Error bars are SD of technical replicates. (c) Flow cytometry of blood samples, gated on B220⁺ cells, showing expression of human Igκ and CGG binding. One WT and one correctly targeted founder mouse are shown. Frequency of Igh^CGG-positive founders in two rounds of injection is shown below (positive/total pups). Numbers within flow plots indicate percentage of cells in the designated gate. (d) Germline transmission of Igh^CGG to F₁ mice. One Igh^CGG founder female was mated to a C57BL6 male, and positive offspring in sequential litters were determined by flow cytometry as in panel c. Frequency of Igh^CGG/+ F₁ pups (positive/total) in each litter is shown below. A Southern blot confirming integration into the Igh locus is also shown. An intact germline Igh locus yields a 14.8-kb fragment, while integration into the locus yields a 8.7-kb fragment.