Gene targeting strategy for B-hIgE/hFCER1A mice. The exons 1-6 of mouse Ighe gene that encode the full-length protein of heavy chain constant region were replaced by human IGHE exon 1-6 in B-hIgE/hFCER1A mice. The exons 1-5 of mouse Fcer1a gene that encode the extracellular domain were replaced by human FCER1A exons 3-7 in B-hIgE/hFCER1A mice.

The blocking effects of Omalizumab analog (in-house) on IgE in mast cells. Bone marrow cells from B-hIgE/hFCER1A mice were cultured with mouse IL-3 protein (PeproTech, 213-13) to differentiate into mast cells and basophils. On day 8, mouse serum was added and co-incubated overnight to assess the binding of human IgE. As shown in Figure A, the serum from B-hIgE/hFCER1A mice, both unstimulated and 4 hours after LPS stimulation, can bind to the mast cells differentiated from B-hIgE/hFCER1A mice with human IgE. In contrast, the serum from C57BL/6JNifdc mice, both unstimulated and 4 hours after LPS stimulation, did not bind to the differentiated mast cells with human IgE. The serum collected 4 hours after LPS stimulation was co-incubated with different doses of Omalizumab analog (in-house) at 37°C for 1 hour, and then added to the cultured cells for co-incubation at 4°C overnight to measure the binding of IgE on the surface of mast cells. As shown in Figure B, Omalizumab analog (in-house) at concentrations of 50 μg/mL and 10 μg/mL demonstrated a significant blocking effect, while some expression of human IgE was detected at 400 ng/m omalizumab analog (in-house). The effect of Omalizumab analog (in-house) at 16 ng/mL was almost negligible.

Strain specific IgE expression analysis in homozygous B-hIgE/hFCER1A mice by flow cytometry. Basophilic granulocytes of the bone marrow were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hIgE/hFCER1A mice (H/H;H/H) stimulated with LPS in vivo, and analyzed by flow cytometry with species-specific anti-IgE antibody. Mouse IgE was detectable in wild-type C57BL/6 mice (+/+). Human IgE was exclusively detectable in homozygous B-hIgE/hFCER1A mice (H/H;H/H) but not in wild-type mice.

Strain specific FCER1A expression analysis in homozygous B-hIgE/hFCER1A mice by flow cytometry. Basophilic granulocytes of the bone marrow were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hIgE/hFCER1A mice (H/H;H/H), and analyzed by flow cytometry with species-specific anti-FCER1A antibody. Mouse FCER1A was detectable in wild-type C57BL/6 mice (+/+). Human FCER1A was exclusively detectable in homozygous B-hIgE/hFCER1A mice (H/H;H/H) but not in wild-type mice (+/+).