Gene targeting strategy for B-hIL31/hIL31RA/hOSMR/hIL4/hIL4RA mice. The mouse Il31 gene that encode the full-length protein were replaced by human IL31 counterpart gene sequences. The coding sequences including human IL31RA extracellular region and mouse Il31ra intracellular region were inserted into mouse IL31RA gene locus. The coding sequences including human OSMR extracellular region and mouse Osmr intracellular region were inserted into mouse Osmr gene locus. The exons 1-4 of mouse Il4 gene that encode the full length coding sequence were replaced by human IL4 exons 1-4. The exons 4-7 of mouse Il4ra gene that encode the extracellular region coding sequences were replaced by human IL4RA exons 4-7.

Strain specific analysis of IL31 mRNA expression in wild-type C57BL/6 mice and B-hIL31/hIL31RA/hOSMR/hIL4/hIL4RA mice by RT-PCR. Testis RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hIL31/hIL31RA/hOSMR/hIL4/hIL4RA mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human IL31 primers. Mouse IL31 mRNA was only detectable in wild-type mice. Human IL31 mRNA was exclusively detectable in homozygous B-hIL31/hIL31RA/hOSMR/hIL4/hIL4RA mice but not in wild-type mice.

Strain specific analysis of IL31RA mRNA expression in wild-type C57BL/6 mice and B-hIL31/hIL31RA/hOSMR/hIL4/hIL4RA mice by RT-PCR. Testis RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hIL31/hIL31RA/hOSMR/hIL4/hIL4RA mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human IL31RA primers. Human IL31RA mRNA was exclusively detectable in homozygous B-hIL31/hIL31RA/hOSMR/hIL4/hIL4RA mice but not in wild-type mice.

Strain specific analysis of OSMR mRNA expression in wild-type C57BL/6 mice and B-hIL31/hIL31RA/hOSMR/hIL4/hIL4RA mice by RT-PCR. Kidney RNA were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hIL31/hIL31RA/hOSMR/hIL4/hIL4RA mice (H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human OSMR primers. Human OSMR mRNA was exclusively detectable in homozygous B-hIL31/hIL31RA/hOSMR/hIL4/hIL4RA mice but not in wild-type mice.

Strain specific IL4 expression analysis in homozygous B-hIL31/hIL31RA/hOSMR/hIL4/hIL4RA mice by ELISA. Serum were collected from wild-type mice C57BL/6 mice (n=3, 6-week-old, female) and homozygous B-hIL31/hIL31RA/hOSMR/hIL4/hIL4RA mice (n=3, 6-week-old, female) that stimulated with anti-mCD3e (7.5 μg/mice, i.p.) and anti-mCD28 (5 μg/mice, i.p.) in vivo for 3 h, and analyzed by ELISA with species-specific IL4 ELISA kit (mIL4 kit: BioLegend 431104; hIL4 kit: BioLegend 430304). Mouse IL4 was only detectable in wild-type mice (A). Human IL4 was exclusively detectable in homozygous mice but not in wild-type mice (B).

Strain specific IL4RA expression analysis in homozygous B-hIL31/hIL31RA/hOSMR/hIL4/hIL4RA mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hIL31/hIL31RA/hOSMR/hIL4/hIL4RA mice stimulated with LPS (200 μg/mice, i.p.) for 2 h, and analyzed by flow cytometry with anti-mouse IL4RA antibody (Biolegend, 144803) or anti-human IL4RA antibody (Biolegend, 355005). Mouse IL4RA was only detectable in wild-type mice, while hIL4RA was exclusively detectable in homozygous mice.