Engineering bNAb-expressing primary mouse B cells. (A) Schematic representation of the targeting strategy to create bNAb-expressing primary mouse B cells. ssDNA HDRT contained 110 nt 5′ and 790 nt 3′ homology arms flanking an expression cassette. The 5′ homology arm is followed by the 111 nt long splice acceptor site and the first two codons of Cμ exon 1, a stop codon, and a SV40 polyadenylation signal (CμSA SV40 pA). Then the mouse Ighv4-9 gene promoter, the leader, and variable and joining regions (VJ) of the respective antibody light chain and mouse κ constant region (C_κ) are followed by a furin-cleavage site, a glycine-serine-glycine (GSG)–linker, and a P2A self-cleaving oligopeptide sequence, the leader, VDJ of the respective antibody heavy chain, and 45 nt of the mouse J_H1 intron splice donor site to splice into downstream constant regions. (B) Experimental setup for C. (C) Flow-cytometric plots of primary, mouse B cells, activated and transfected with RNPs targeting the Ighj4 intron and Igkc exon with or without ssDNA HDRTs encoding the 3BNC60^SI, 3BNC117, or 10-1074 antibody. Non-transfected, antigen-binding B cells from 3BNC60^SI knock-in mice cultured the same way are used as control for gating. (D) Quantification of C. Each dot represents one transfection. Data from seven independent experiments (B–D). (E) Experimental setup for F–H. (F) Flow-cytometric plots of primary, mouse B cells, activated and transfected using ssDNA HDRT encoding the antibodies 3BNC60^SI, 3BNC117, PGT121, or 10-1074. B cells were expanded on feeder cells for 3 d. Cultured, nontransfected, antigen-binding B cells from PGT121 knock-in mice are shown for gating. (G) Quantification of F. (H) Total number of antigen-binding B cells before (24 h) or after 3 d (day 4) of feeder culture. Bars indicate mean ± SEM. Combined data from two independent experiments for E–H.