Gene targeting strategy for B-hIL4/hIL4RA plus mice(C). The exons 1-4 of mouse Il4 gene that encode the full length coding sequence were replaced by human IL4 exons 1-4 in B-hIL4/hIL4RA plus mice(C). The exons 4-11 of mouse Il4ra gene that encode the extracellular domain, transmembrane domain, cytoplasmic region and 3’UTR are replaced by human counterparts in B-hIL4/hIL4RA plus mice(C). The promoter and 5’UTR region of the mouse gene are retained.

Strain specific IL4RA expression analysis in wild-type BALB/cCrSlcNifdc mice and homozygous B-hIL4/hIL4RA plus mice(C) by flow cytometry. Splenocytes were collected from wild-type BALB/cCrSlcNifdc (+/+) and homozygous B-hIL4/hIL4RA plus mice(C) (H/H,H/H) stimulated with anti-CD3ε (BioXcell, BE0001) in vivo for 24 hrs, and protein expression was analyzed with anti-mouse IL4RA antibody (Biolegend, 144804) and anti-human IL4RA antibody (Biolegend, 355006) by flow cytometry. Mouse IL4RA was detectable in wild-type BALB/cCrSlcNifdc mice. Human IL4RA was detectable in homozygous B-hIL4/hIL4RA plus mice(C), but not in wild-type mice. WT: wild-type C57BL/6JNifdc mice; HO: homozygous B-hIL4/hIL4RA plus mice(C).

Function analysis of IL4RA in homozygous B-hIL4/hIL4RA plus mice(C). Splenocytes were collected from wild-type BALB/cCrSlcNifdc and homozygous B-hIL4/hIL4RA plus mice(C). Splenic B cells were separated via Pan B Cell Isolation Kit (Miltenyi, 130-104-443) and then cultured in 96-well plates stimulated with mCD40L, hIL4 (Sino Biological, GMP-11846-HNAE) or mIL4 (Miltenyi, 130-097-760) for 6 days. Then cell culture supernatants were collected for ELISA analysis of mouse IgE by kit (BioLegend, 432404). Mouse IgE was exclusively induced in wild-type BALB/cCrSlcNifdc mice stimulated with mIL4 and homozygous mice stimulated with hIL4, indicating that hIL4RA functions well in homozygous mice.