Basic Information

Strain name
C57BL/6-Osmtm1(OSM)BcgenOsmrtm1(OSMR)Bcgen/Bcgen
Common Name
B-hOSM/hOSMR mice
Stock Number
121329
Background
C57BL/6
Aliases
OSMR: OSMRB; PLCA1; IL-31RB; OSMRbeta; IL-31R-beta
NCBI Gene ID

Targeting strategy

Gene targeting strategy for B-hOSM/hOSMR mice.

The mouse Osm gene that encode the full-length protein were replaced by human OSM counterpart gene sequences in B-hOSM/hOSMR mice. The coding sequences including human OSMR extracellular region and mouse Osmr intracellular region were inserted into mouse Osmr gene in B-hOSM/hOSMR mice.

mRNA and protein expression analysis

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. (B) Protein expression. The OSMR protein were detected both in wild-type mice and homozygous B-hOSM/hOSMR mice. 293T cells were used as positive control (PC).

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.

Human OSM induced IL-6 secretion in MEFs

Human OSM 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.

Leukocyte analysis

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).

Summary

mRNA expression analysis:

Human OSMR mRNA was detectable in B-hOSM/hOSMR mice but not in wild-type mice.

Protein expression analysis:

Human OSM and OSMR were detectable in B-hOSM/hOSMR mice.

Function analysis:

hOSM induced IL-6 secretion in MEFs of homozygous B-hOSM/hOSMR mice, and the function is blockade by anti-hOSMR antibody. The results show that OSMR signaling function was not influenced by gene humanization.

Leukocytes cell subpopulation analysis

Target gene humanization does not change the overall development, differentiation or distribution of leukocytes in spleen, blood and lymph node.

Back to top