• TL1A binds to death receptor 3 (DR3) to provide stimulatory signals for downstream signaling pathways, thereby regulating the proliferation, activation, apoptosis of effector cells, and the production of cytokines and chemokines. Soluble decoy receptor 3 (DcR3) may neutralize the effects of sTL1A/DR3. In addition, DcR3 can inhibit apoptosis, reduce inflammation, and prevent tissue damage by neutralizing LIGHT and FasL.
• IL-23 is a heterodimeric cytokine composed of p40 and p19 subunits, primarily produced by macrophages and dendritic cells. IL-23 binds to its receptor IL-23R, which regulates the release of downstream pro-inflammatory cytokines.
• The exons 1-4 of mouse Tl1a gene that encode extracellular domain were replaced by human counterparts in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. The genome of the mouse Il23a gene encoding the full-length protein was replaced with human IL23A counterpart in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. The genome of the mouse Il12b gene encoding the full-length protein was replaced with human IL12B counterpart in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. The exon 3 and 3’UTR region of mouse Rag2 were knocked out in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice, resulting in a disruption of the Rag2 gene.
• Human TL1A and IL23 protein were only detectable in homozygous B-hTL1A/hIL23A/hIL12B, Rag2 KO mice.
• Frequency of various immune cells including T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Treg cells in spleen and blood of B-hTL1A/hIL23A/hIL12B, Rag2 KO mice were similar to those in B-Rag2 KO mice.
• This product is used for the evaluation of the pharmacodynamics and safety of anti-human TL1A and IL23 antibodies in autoimmune diseases such as inflammatory bowel disease.

Gene targeting strategy for B-hTL1A/hIL23A/hIL12B, Rag2 KO mice.

The exons 1-4 of mouse Tl1a gene that encode extracellular domain were replaced by human counterparts in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. The genomic region of mouse Tl1a gene that encodes transmembrane domain and cytoplasmic portion was retained. The promoter, 5’UTR and 3’UTR region of the mouse gene were also retained. The chimeric TL1A expression was driven by endogenous mouse Tl1a promoter, while mouse Tl1a gene transcription and translation will be disrupted.

The exons 1-4 of mouse Il23a gene that encode the whole molecule (ATG to STOP codon) were replaced by human counterparts in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. The promoter, 5’UTR and 3’UTR region of the mouse gene were retained. The human IL23A expression was driven by endogenous mouse Il23a promoter, while mouse Il23a gene transcription and translation will be disrupted.

The exons 2-8 of mouse Il12b gene that encode the whole molecule (ATG to STOP codon), including 3’UTR were replaced by human counterparts in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. The promoter and 5’UTR region of the mouse gene were retained. The human IL12B expression was driven by endogenous mouse Il12b promoter, while mouse Il12b gene transcription and translation will be disrupted.

The exon 3 and 3’UTR region of mouse Rag2 were knocked out in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice, resulting in a disruption of the Rag2 gene.

Soluble TL1A expression analysis in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice by ELISA. Bone marrow derived dendritic cells (BMDCs) were produced by culturing the bone marrow from wild-type C57BL/6JNifdc mice (+/+), and homozygous B-hTL1A/hIL23A/hIL12B, Rag2 KO mice (H/H;H/H;H/H;-/-), which were stimulated with 1 μg/mL LPS in vitro for 24 hrs. After stimulation, the supernatants were collected and the levels of soluble TL1A were measured using a species-specific human TL1A ELISA kit (R&D, DY1319-05). Soluble human TL1A was exclusively detectable in homozygous B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. Values are expressed as mean ± SEM. ND: not detectable.

Mouse IL-23 and human IL-23 expression analysis in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice by ELISA. Bone marrow derived dendritic cells were produced by culturing the bone marrow from wild-type C57BL/6JNifdc mice (+/+), and homozygous B-hTL1A/hIL23A/hIL12B, Rag2 KO mice (H/H;H/H;H/H;-/-), which were stimulated with 1 μg/mL LPS in vitro for 24 hrs. After stimulation, the supernatants were collected and the levels of mouse and human IL23 were analyzed by ELISA(R&D, M2300; R&D, D2300B). Mouse IL23 was only detectable in wild-type C57BL/6JNifdc mice. Human IL23 was exclusively detectable in homozygous B-hTL1A/hIL23A/hIL12B, Rag2 KO mice. Values are expressed as mean ± SEM. ND: not detectable.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6JNifdc mice (female, n=3, 8-week-old), B-Rag2 KO mice (female, n=3, 8-week-old) and B-hTL1A/hIL23A/hIL12B, Rag2 KO mice (female, n=3, 8-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice were similar to those in B-Rag2 KO mice. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Blood was isolated from wild-type C57BL/6JNifdc mice (female, n=3, 8-week-old), B-Rag2 KO mice (female, n=3, 8-week-old) and B-hTL1A/hIL23A/hIL12B, Rag2 KO mice (female, n=3, 6-week-old). A. Flow cytometry analysis of the blood was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Percentages of T cells, B cells, NK cells, dendritic cells, neutrophils, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in B-hTL1A/hIL23A/hIL12B, Rag2 KO mice were similar to those in B-Rag2 KO mice. Values are expressed as mean ± SEM.