Basic Information
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Targeting strategy
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Gene targeting strategy for B-hIL31/hIL31RA/hOSM/hOSMR mice.
- The mouse Il31 gene that encode the full-length protein were replaced by human IL31 counterpart gene sequences in B-hIL31/hIL31RA/hOSM/hOSMR mice.
- The mouse Osm gene that encode the full-length protein were replaced by human OSM counterpart gene sequences in B-hIL31/hIL31RA/hOSM/hOSMR mice.
- The coding sequences including human IL31RA extracellular region and mouse Il31ra intracellular region were inserted into mouse IL31RA gene in B-hIL31/hIL31RA/hOSM/hOSMR mice.
- The coding sequences including human OSMR extracellular region and mouse Osmr intracellular region were inserted into mouse Osmr gene in B-hIL31/hIL31RA/hOSM/hOSMR mice.
Note: Mice were obtained by B-hIL31/hIL31RA mice cross breeding with B-hOSM/hOSMR mice.
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mRNA expression analysis
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Strain specific analysis of IL31 and IL31RA gene expression in wild-type C57BL/6 mice and homozygous B-hIL31/hIL31RA mice by RT-PCR. Testis was collect from wild-type mice (+/+) and B-hIL31/hIL31RA mice (H/H). Mouse Il31 and Il31ra mRNA were exclusively detectable in wild-type mice. Human IL31 and IL31RA mRNA were exclusively detectable in homozygous B-hIL31/hIL31RA mice, but not in wild-type mice.
Strain specific analysis of OSMR gene expression in wild-type mice and B-hOSM/hOSMR mice. Kidney was collected from wild-type C57BL/6 mice (+/+) and homozygous B-hOSM/hOSMR mice (H/H), and analyzed by RT-PCR and western blot. (A) mRNA expression. Mouse Osmr mRNA was exclusively detectable in wild-type mice. Human OSMR mRNA was exclusively detectable in homozygous B-hOSM/hOSMR mice.
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Protein expression analysis
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Strain specific OSM expression analysis in homozygous B-hOSM/hOSMR mice by ELISA. Spleen were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hOSM/hOSMR mice (H/H), and stimulated with anti-mouse CD3 and anti-mouse CD28 antibodies in vitro. Cell culture supernatant were collected and analyzed by species-specific OSM ELISA kit. Mouse OSM was detectable in wild-type mice. Human OSM was exclusively detectable in homozygous B-hOSM/hOSMR mice but not in wild-type mice.
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hOSM induced IL-6 secretion in MEFs of B-hOSM/hOSMR mice
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hOSM induced IL-6 secretion is blockade by anti-hOSMR antibody in MEFs of homozygous B-hOSM/hOSMR mice. Mouse embryonic fibroblasts (MEFs) were derived from either C57BL/6 mice or B-hOSM/hOSMR mice, and stimulated with different concentrations of mOSM (0-100ng/mL) or hOSM (0-1000ng/mL) for 24h. Supernatants from each well were collected and analyzed for IL-6 expression by ELISA. (A) In the MEFs of C57BL/6 mice, IL-6 can be induced by mOSM in a dose-dependent manner, but not by hOSM. It demonstrates that hOSM can’t be recognized by mouse receptor. (B) In the MEFs of B-hOSM/hOSMR mice, hOSM can induce IL-6 secretion, and the function can be blocked by anti-hOSMR antibodies (in house). The results show that the OSMR signaling function was not influenced by gene humanization. Cells were incubated with antibodies (10μg/mL) for 30 minutes, and stimulated with hOSM.
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hIL31 induces pruritus in B-hIL31/hIL31RA/hOSMR mice
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hIL31 induces pruritus in B-hIL31/hIL31RA/hOSMR mice. hIL-31 are administered to homozygous B-hIL31/hIL31RA/hOSMR mice (female, n=3) on Day0, Day3, Day7 and Day10. Meanwhile, recording the scratching counts for 3 hours from injection of hIL-31, and scratching counts before hIL-31 injection as the baseline. The data were shown as the delta scratching counts, which equal to scratching counts in 3-hour subtract baseline. As shown in the panel, hIL-31 induced pruritus successfully in B-hIL31/hIL31RA/hOSMR mice. An antibody targeting IL-31 signaling was used as positive control, which showed a significant antipruritic effect in our model. The data was provided by a cooperative partner.
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Immune cell analysis
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Analysis of spleen leukocyte subpopulations by flow cytometry. Splenocytes were isolated from C57BL/6, B-hOSM/hOSMR mice, B-hIL31/hIL31RA/hOSMR mice and B-hIL31/hIL31RA/hOSM/hOSMR mice (n=3, 6 or 7-week-old, female, homozygous). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. (A) Spleen leukocyte subpopulations. (B) T cells subpopulations. Percent of T cells (CD8+ T cells, CD4+ T cells, and Tregs), B cells, NK cells, dendritic cells, neutrophils, macrophages and monocytes in B-hOSM/hOSMR mice, B-hIL31/hIL31RA/hOSMR mice and B-hIL31/hIL31RA/hOSM/hOSMR mice were similar to those in the C57BL/6 mice. Data was shown as mean ± SD, and analyzed using Ordinary one-way ANOVA method. (*p<0.05).
Analysis of blood leukocyte subpopulations by flow cytometry. Blood cells were isolated from C57BL/6, B-hOSM/hOSMR mice, B-hIL31/hIL31RA/hOSMR mice and B-hIL31/hIL31RA/hOSM/hOSMR mice (n=3, 6 or 7-week-old, female, homozygous). Flow cytometry analysis of the blood was performed to assess leukocyte subpopulations. (A) Blood leukocyte subpopulations. (B) T cells subpopulations. Percent of T cells (CD8+ T cells, CD4+ T cells, and Tregs), B cells, NK cells, dendritic cells, neutrophils, macrophages and monocytes in B-hOSM/hOSMR mice, B-hIL31/hIL31RA/hOSMR mice and B-hIL31/hIL31RA/hOSM/hOSMR mice were similar to those in the C57BL/6 mice. Data was shown as mean ± SD, and analyzed using Ordinary one-way ANOVA method. (*p<0.05).
Analysis of lymph node leukocyte subpopulations by flow cytometry. Lymph node cells were isolated from C57BL/6, B-hOSM/hOSMR mice, B-hIL31/hIL31RA/hOSMR mice and B-hIL31/hIL31RA/hOSM/hOSMR mice (n=3, 6 or 7-week-old, female, homozygous). Flow cytometry analysis of the lymph node was performed to assess leukocyte subpopulations. (A) Lymph node leukocyte subpopulations. (B) T cells subpopulations. Percent of T cells (CD8+ T cells, CD4+ T cells, and Tregs), B cells and NK cells in B-hOSM/hOSMR mice, B-hIL31/hIL31RA/hOSMR mice and B-hIL31/hIL31RA/hOSM/hOSMR mice were similar to those in the C57BL/6 mice. Data was shown as mean ± SD, and analyzed using Ordinary one-way ANOVA method. (*p<0.05).
