IL-12 and IL-23 play a crucial role in various immune-mediated diseases, including inflammatory bowel disease, plaque psoriasis, and rheumatoid arthritis.

• Gene Information: IL-12 is a heterodimer of p40 and p35 subunits, while IL-23 is composed of p19 and p40. Correspondingly, IL-12 signals through the IL-12Rβ1/IL-12Rβ2 complex, whereas IL-23 utilizes a receptor pair consisting of IL-12Rβ1 and IL-23R.
• Protein Expression: The expression of these two cytokines is tightly regulated and occurs primarily in antigen-presenting cells (such as dendritic cells and macrophages) as part of the response to pathogen stimulation.
• Signaling Pathway: IL-12 stimulation of JAK2 and TYK2 activity leads to phosphorylation of STAT4 and other STAT molecules. IL-23 also activates the JAK-STAT pathway but acts mainly on STAT3. IL-12 could induce the production of IFN-γ, which is required for the development of TH1 immune response. IL-23 could induce IL-17A, IL-17F and/or IL-22 and stabilizes TH17 cells.
• Therapeutic Inhibition: By inhibiting IL-12 and IL-23 signaling, these inhibitors suppress pro-inflammatory cytokine production and modulate the immune response to alleviate clinical symptoms.

IL23A

• The exons 1-4 of mouse Il23a gene that encode the full length of encoding regions were replaced by human IL23A counterpart gene sequences.
• The promoter, 5’UTR and 3’UTR region of the mouse gene were also retained. The human IL23A expression was driven by endogenous mouse Il23a promoter, while mouse Il23a gene transcription and translation will be disrupted.

IL12B

• 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.
• The promoter and 5’UTR region of the mouse gene were also retained. The human IL12B expression was driven by mouse Il12b promoter, while mouse IL12B  gene transcription and translation will be disrupted.

IL12A

• The exons 1-7 of mouse Il12a gene that encode the full length of encoding regions were replaced by human IL12A counterpart gene sequences.
• The promoter, 5’UTR and 3’UTR region of the mouse gene were also retained. The human IL12A expression was driven by endogenous mouse Il12a promoter, while mouse Il12a gene transcription and translation will be disrupted.

IL23R

• A chimeric CDS that encodes mouse IL23R signal peptide, human IL23R extracellular domain, mouse IL23R transmembrane and cytoplasmic domain, followed by mouse 3’UTR-STOP was inserted after exon 3 of mouse Il23r.
• The chimeric IL23R protein expression will be driven by endogenous mouse Il23r promoter, while mouse IL23r gene transcription and translation will be disrupted.

IL12RB1

• The exons 1-14 of mouse Il12rb1 gene that encode signal peptide and extracellular domain were replaced by human counterparts. The genomic region of mouse Il12rb1 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 human IL12RB1 expression was driven by endogenous mouse Il12rb1 promoter, while mouse Il12rb1 gene transcription and translation will be disrupted.

IL12RB2

• A chimeric CDS that encodes mouse IL12RB2 signal peptide, human IL12RB2 extracellular domain, mouse IL12RB2 transmembrane and cytoplasmic domain, followed by mouse 3’UTR-STOP was inserted after exon 2 of mouse Il12rb2.
• The chimeric IL12RB2 protein expression will be driven by endogenous mouse Il12rb2 promoter.

• Mouse Il23a, Il12a, Il12b, Il23r, Il12rb1, Il12rb2 mRNA were detectable in wild-type mice. 
• Human IL23A, IL12A, IL12B, IL23R, IL12RB1, IL12RB2 mRNA was detectable in homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1/hIL12RB2 mice.

Strain specific analysis of IL23A, IL12A, IL12B, IL23R, IL12RB1, IL12RB2 mRNA expression in wild-type C57BL/6JNifdc mice and homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1/hIL12RB2 mice by RT-PCR. Spleen RNA was isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1/hIL12RB2 mice (H/H, H/H, H/H, H/H , H/H, H/H), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human IL23A, IL12A, IL12B, IL23R, IL12RB1, IL12RB2 primers. Mouse Il23a, Il12a, Il12b, Il23r, Il12rb1, Il12rb2 mRNA were detectable in wild-type mice. Human IL23A, IL12A, IL12B, IL23R, IL12RB1, IL12RB2 mRNA was detectable in homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1/hIL12RB2 mice.

• Mouse IL23 was only detectable in wild-type C57BL/6 mice after LPS stimulation. 
• Human IL23 was exclusively detectable in homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice after LPS stimulation.

Strain specific IL23 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice by ELISA. Bone marrow-derived dendritic cells were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice (H/H, H/H, H/H, H/H , H/H, H/H) stimulated with LPS in vitro for 24 hrs, then cell supernatants were collected and analyzed by ELISA (Mouse IL-23 Quantikine ELISA Kit, R&D, M2300; Human IL-23 Quantikine ELISA Kit, R&D, D2300B). Mouse IL23 was only detectable in wild-type C57BL/6 mice and human IL23 was exclusively detectable in homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice after LPS stimulation. Values are expressed as mean ± SEM.

• Mouse IL12 was only detectable in wild-type C57BL/6JNifdc mice after LPS stimulation. 
• Human IL12 was exclusively detectable in homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice after LPS stimulation.

Strain specific IL12 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice by ELISA. Bone marrow-derived dendritic cells were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice (H/H, H/H, H/H, H/H, H/H, H/H) stimulated with LPS in vitro for 24 hrs, then cell supernatants were collected and analyzed by ELISA (Mouse IL-12(p70) ELISA MAXTM Deluxe Set , Biolegend, 433607; Human IL12 p70 SimpleStep ELISA kit, Abcam, ab223592). Mouse IL12 was only detectable in wild-type C57BL/6JNifdc mice and human IL12 was exclusively detectable in homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice after LPS stimulation. Values are expressed as mean ± SEM.

• Expression of mouse IFNγ was increased in wild-type C57BL/6JNifdc mice and homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice after mIL12, hIL12 stimulation. 
• Humanized IL12A, IL12B, IL12RB1, and IL12RB2 are able to perform their functions normally.

Strain specific IFNγ expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice by ELISA. Spleen CD4+ T cells supernatant was collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice (H/H, H/H, H/H, H/H, H/H, H/H) stimulated with  mIL12, hIL12 in vitro for 48 hrs. Expression level mouse IFNγ were analyzed by ELISA (Mouse IFNγ ELISA MAXTM Deluxe Set, 430804). Expression of mouse IFNγ was increased in wild-type C57BL/6JNifdc mice and homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice after mIL12, hIL12 stimulation. Values are expressed as mean ± SEM.

• Expression of mouse IL17A was increased in wild-type C57BL/6JNifdc mice and homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice after mIL23, hIL23 stimulation. 
• Humanized IL23A, IL12B, IL23R, and IL12RB1 mice are able to perform their functions, although their reactivity is lower compared with that of wild-type C57 mice.

Strain specific IL17A expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice by ELISA. Spleen CD4+ T cells supernatant was collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice (H/H, H/H, H/H, H/H, H/H, H/H) stimulated with  mIL23, hIL23 in vitro for 48 hrs. Expression level mouse IL17A were analyzed by ELISA (Mouse IL-17A ELISA Kit, KE10020). Expression of mouse IL17A was increased in wild-type C57BL/6JNifdc mice and homozygous B-hIL23A/hIL12B/hIL12A/hIL23R/hIL12RB1 plus/hIL12RB2 ad mice after mIL23, hIL23 stimulation. Values are expressed as mean ± SEM.