Generation and validation of conditional hMYD88(L265P)^LSLtransgenic mouse model. (A) Schematic representation of the targeting vector integration site downstream of the mouse Col1A1 gene that supports high transgene expression in a variety of cell types including B cells. WT or L265P-mutated human MYD88 complementary DNA (cDNA) sequences were cloned into the targeting vector downstream of the loxP-flanked stop cassette (LSL) and electroporated together with an Flp recombinase-expressing plasmid into the C2 mouse embryonic stem cell line. C2 cells are engineered to acquire hygromycin resistance after targeted integration of the gene of interest at flippase recognition target (frt) sites. (B) Sanger sequencing of the human MYD88 sequence amplified from splenic genomic DNA isolated from transgenic mice. The presence of T>C transition results in the L265P mutation in MYD88^L265P mice. (C) AID^Cre-induced deletion of the LSL cassette assessed by polymerase chain reaction (PCR) using primers indicated as arrows in panel A in FACS splenic T cells and GC B cells from MYD88^WT (n = 3) and MYD88^L265P (n = 3) mice immunized with sheep red blood cells (SRBCs). loxP-flanked sequence: 640 bp; deleted: 330 bp. (D) Immunoblot analysis of MYD88 protein expression and p65 (S534) phosphorylation in FACS splenic GC B cells from AID^Cre (n = 2), MYD88^WT (n = 2), and MYD88^L265P (n = 2) mice immunized with SRBCs relative to actin. (E) Immunohistochemical analysis of MYD88 expression within GCs of AID^Cre, MYD88^WT, and MYD88^L265P mice. GCs were identified based on morphology and the presence of apoptotic bodies as well as Ki-67 staining in serial consecutive sections. Note punctate, cytoplasmic MYD88 staining in the MYD88^L265P (white arrowheads) but not MYD88^WT or AID^Cre mice. Scale bars, 20 µm (see also supplemental Figure 1A). (F) Immunofluorescence analysis of p65 (white) subcellular localization within GC B cells (peanut agglutinin [PNA]; green) in spleens from AID^Cre, MYD88^WT, and MYD88^L265P mice immunized with SRBCs (top and middle). Lower-magnification images (×40) were quantified using ImageJ (bottom). Gates were set up to quantify voxels with high intensity of p65 and low intensity of PNA staining or high intensity of PNA and low intensity of p65 staining. The numbers represent mean intensity of voxels within the gated region, which is proportional to the number of events gated. Note increased nuclear p65 staining (quantified as voxels positive for p65 and negative for PNA, which stain cell membrane) in the MYD88^WT and MYD88^L265P but not AID^Cre mice. Scale bars, 20 µm (see also supplemental Figure 1B-C). (G) Spleen weights in 8- to 16-week-old AID^Cre, MYD88^WT, and MYD88^L265P mice (n = 3 per group). Graphs depict the mean ± standard deviation (SD). P values were calculated by using Welch’s t test (left). Representative gross pictures of spleens (Sp) and LNs are shown on the right. Scale bars, 1 cm. Histologic (hematoxylin and eosin [H&E]) and IHC stains of indicated cell markers on consecutive serial sections from spleens (H) and BMs (I) from representative 8- to 16-week-old AID^Cre, MYD88^WT, and MYD88^L265P mice. B220, B cells; CD3, T cells; CD138, plasma cells. Scale bars: white, 100 µm; black, 25 µm. pA, polyadenylation sequences; CAG, cytomegalovirus enhancer/chicken β-actin promoter; PGK ATGfrt, phosphoglycerate kinase 1 promoter followed by ATG initiation codon restoring hygromycin resistance gene expression.