Targeting strategy

Gene targeting strategy for B-hIL23A/hIL12B mice. The exons 1-4 of mouse Il23a gene that encode the full length of encoding regions were replaced by human IL23A counterpart gene sequences in B-hIL23A/hIL12B mice. The exons 2-8 of mouse Il12b gene that encode the full length of encoding regions and 3’UTR were replaced by human IL12B counterpart gene sequences in B-hIL23A/hIL12B mice.

mRNA expression analysis

Strain specific analysis of IL23A and IL12B gene expression in B-hIL23A/hIL12B mice by RT-PCR. Mouse Il23a and Il12b mRNA were detectable in thymocyte of wild-type mice (+/+) . Human IL23A and IL12B mRNA were detectable only in homozygous B-hIL23A/hIL12B mice (H/H) but not in wild-type C57BL/6 mice. 

Strain specific IL23 (IL23A/IL12B) expression analysis in wild-type C57BL/6 mice, homozygous B-hIL23A/hIL12B mice and B-hTL1A/hIL23A/hIL12B mice by ELISA. Bone marrow derived dendritic cells were produced by culturing the bone marrow from wild-type C57BL/6 mice, homozygous B-hIL23A/hIL12B mice and B-hTL1A/hIL23A/hIL12B mice (female, 6-week-old, n=3), which were stimulated with 10 μg/mL LPS in vivo for 24 h . After stimulation, the supernatants were collected and the levels of mouse IL23 and human IL23 were analyzed by ELISA. Mouse IL23 was only detectable in wild-type C57BL/6 mice. Human IL23 was exclusively detectable in homozygous B-hIL23A/hIL12B mice and B-hTL1A/hIL23A/hIL12B mice. Values are expressed as mean ± SEM. ND: not detectable.

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hIL23A/hIL12B mice (n=3, 7-week-old). Flow cytometry analysis of the splenocytes were performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hIL23A/hIL12B mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL23A and hIL12B in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen.

Analysis of subpopulation of T cells in lymph node by FACS. Lymph nodes were isolated from female C57BL/6 and B-hIL23A/hIL12B mice (n=3, 7-week-old). Flow cytometry analysis of the lymph nodes were performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ T cells were gated for TCRβ+ cells population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD4+ T cells, CD8+ T cells and Treg cells in homozygous B-hIL23A/hIL12B mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hIL23A and hIL12B in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in lymph node. Values are expressed as mean ± SEM.

Hematology analysis

Complete blood count (CBC) of B-hIL23A/hIL12B mice. Values are expressed as mean ± SD.

Biochemistry analysis

Biochemical test of B-hIL23A/hIL12B mice. Values are expressed as mean ± SD.

Effects of anti-human IL23A antibody in psoriasis-like skin lesions model of B-hIL23A/hIL12B mice. (A) Body weight changes during treatment. (B-C) Erythema and the scaling score of the back were scored daily on a scale from 0 to 4. Additionally, the cumulative score (erythema plus scaling) is depicted (D). Results indicated that structural features of IMQ-induced skin inflammation is visible and increased in severity up to day 5. Anti-human IL23A antibody can efficiently decrease the development of the psoriasis-like disease in B-hIL23A/hIL12B mice.