• 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 soluble TL1A (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 genome of the mouse Tl1a gene encoding the extracellular domain was replaced with human TL1A counterpart in B-hTL1A/hDR3/hIL23A/hIL12B mice. The genome of the mouse DR3 gene encoding the full-length protein was replaced with human DR3 counterpart in B-hTL1A/hDR3/hIL23A/hIL12B mice. The genome of the mouse Il23a gene encoding the full-length protein was replaced with human IL23A counterpart in B-hTL1A/hDR3/hIL23A/hIL12B mice. The genome of the mouse Il12b gene encoding the full-length protein was replaced with human IL12B counterpart in B-hTL1A/hDR3/hIL23A/hIL12B mice.
• Mouse Tl1a, DR3, Il23a, and Il12b mRNA were detectable in wild-type C57BL/6JNifdc mice, and human TL1A, DR3, IL23A, and IL12B mRNA were exclusively detectable in homozygous B-hTL1A/hDR3/hIL23A/hIL12B mice.
• Human TL1A, DR3, IL23 protein were exclusively detectable in homozygous B-hTL1A/hDR3/hIL23A/hIL12B mice.
• This product is used for the evaluation of the pharmacodynamics and safety of anti-human TL1A/DR3/IL23 antibodies in autoimmune diseases such as inflammatory bowel disease.

Gene targeting strategy for B-hTL1A/hDR3/hIL23A/hIL12B mice.

The exons 1-4 of mouse Tl1a gene that encode extracellular domain were replaced by human counterparts in B-hTL1A/hDR3/hIL23A/hIL12B 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 1-10 of mouse DR3 gene that encode the whole molecule (ATG to STOP codon), including promoter, 5’UTR and 3’UTR were replaced by human counterparts in B-hTL1A/hDR3/hIL23A/hIL12B mice. The human DR3 expression was driven by human DR3 promoter, while mouse DR3 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/hDR3/hIL23A/hIL12B 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/hDR3/hIL23A/hIL12B 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.

Strain specific analysis of TL1A/DR3/IL23A/IL12B mRNA expression in wild-type C57BL/6JNifdc mice and B-hTL1A/hDR3/hIL23A/hIL12B mice by RT-PCR. Spleen, lung and colon RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hDR3/hIL23A/hIL12B mice (H/H;H/H;H/H;H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human TL1A/DR3/IL23A/IL12B primers. Mouse Tl1a, DR3, Il23a, and Il12b mRNA were detectable only in wild-type C57BL/6JNifdc mice. Human TL1A, DR3, IL23A, and IL12B mRNA were detectable only in homozygous B-hTL1A/hDR3/hIL23A /hIL12B mice but not in wild-type C57BL/6JNifdc mice.

Strain specific TL1A expression analysis in homozygous B-hTL1A/hDR3/hIL23A/hIL12B mice by ELISA. Bone marrow-derived dendritic cells were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hDR3/hIL23A /hIL12B mice(H/H;H/H;H/H;H/H) (female, 7-week-old, n=3), and stimulated with 1 μg/mL LPS  in vitro for 24 h, then cell supernatants were collected and analyzed by ELISA (anti-human TL1A ELISA kit: R&D, DY1319-05). Human TL1 was exclusively detectable in homozygous B-hTL1A/hDR3/hIL23A /hIL12B mice but not in wild-type mice. Values are expressed as mean ± SEM. ND: not detectable.

Strain specific DR3 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hTL1A/hDR3/hIL23A/hIL12B mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hDR3/hIL23A/hIL12B mice (H/H;H/H;H/H;H/H), protein expression was analyzed with anti-mouse DR3 antibody (Biolegend, 144407) and anti-human DR3 antibody (Biolegend, 307105) by flow cytometry. Mouse DR3 was detectable on Treg cells of wild-type C57BL/6JNifdc mice, human DR3 was detectable on Treg cells of homozygous B-hTL1A/hDR3/hIL23A/hIL12B mice.

Strain specific TNFRSF25 expression analysis in in wild-type C57BL/6JNifdc mice and homozygous B-hTL1A/hDR3/hIL23A/hIL12B mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTL1A/hDR3/hIL23A/hIL12B mice (H/H;H/H;H/H;H/H), and analyzed by flow cytometry with anti-human DR3 antibody Ab1, which is offered by the client. Ab1 binding were only detected on Treg cells of homozygous B-hTL1A/hDR3/hIL23A/hIL12B mice.

Mouse IL-23 and human IL-23 expression analysis in B-hTL1A/hDR3/hIL23A /hIL12B 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/hDR3/hIL23A /hIL12B mice(H/H;H/H;H/H;H/H) (female, 7-week-old, n=3), which were stimulated with 1 μg/mL LPS in vitro for 24h. After stimulation, the supernatants were collected and the levels of mouse and human IL23 were analyzed by ELISA. Mouse IL23 was only detectable in wild-type C57BL/6JNifdc mice. Human IL23 was exclusively detectable in homozygous B-hTL1A/hDR3/hIL23A /hIL12B mice. Values are expressed as mean ± SEM. ND: not detectable.