• Beta 2-microglobulin (β2M) is a 12 kDa, Ig like, non-membrane-anchored glycoprotein. It non-covalently associates with MHC class I heavy chain and some Class I-like molecules and serves as the light chain. In association with MHC class I, β2M is expressed on all leukocytes, platelets, endothelial cells, and epithelial cells. β2M plays an essential role in stabilizing MHC class I complexes for antigen presentation.
• H2-Ab1 gene encoding the beta-chain of the Class II heterodimer H2-A. Enables several functions, including peptide antigen binding activity; protein antigen binding activity; and toxic substance binding activity. Involved in several processes, including B cell affinity maturation; cellular response to interferon-gamma; and positive regulation of T-helper 1 type immune response. Acts upstream of or within antigen processing and presentation of exogenous peptide antigen via MHC class II and immune response.
• Mesothelin, also known as CAK1 and ERC, is derived from a precursor that also includes Megakaryocyte Potentiating Factor (MPF). Following cleavage of the precursor, Mesothelin remains attached to the cell surface via a GPI linkage.
• B7-H3 may play a dual role in the immune system. On the one hand, B7-H3 acts as a co-stimulatory molecule that co-stimulates CD4+ and CD8+ cells, induces cellular immunity and selectively enhances interferon-γ (IFNG) production in response to T cell receptor signaling. On the other hand, B7-H3 also has a co-inhibitory effect, which inhibits Treg cells, thereby allowing tumors to escape the immune response. Antibodies targeting B7-H3 inhibit its immunosuppressive effects, suppressing and killing tumors.
• The murineB2mandH2-Ab1gene were knocked out while a fused gene composed of murineB2mandFcgrtgene was inserted after the signal peptide sequence of murineFcgrtgene in B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice. Exons 11-17 of the mouseMslngene that encode the full-length protein were replaced by humanMSLNexons 11-18 of the B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice. The exons 3~4 of mouseCd276gene that encode the extracellular domain were replaced by humanCD276exons 3~6 in B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice.
• MHC I and MHC II were only detectable in wild-type B-NDG mice, but not in homozygous B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice.
• Mouse MSLN was detected in lung and ovary in wild-type mice, human MSLN was detected in lung and ovary in homozygous B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice. Human B7-H3 was detected in lung, liver, stomach, brain and heart in homozygous B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice.

Gene targeting strategy for B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice.
The murineB2mandH2-Ab1gene were knocked out while a fused gene composed of murineB2mandFcgrtgene was inserted after the signal peptide sequence of murineFcgrtgene in B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice.
Exons 11-17 of the mouseMslngene that encode the full-length protein were replaced by humanMSLNexons 11-18 of the B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice. The promoter, 5’ UTR and 3’UTR region of the mouse gene are retained. The human MSLN expression is driven by endogenous mouseMslnpromoter, while mouse Msln gene transcription and translation will be disrupted.
The exons 3~4 of mouseCd276gene that encode the extracellular domain were replaced by humanCD276exons 3~6 in B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice. The promoter, 5’ UTR and 3’UTR region of the mouse gene are retained. The human CD276 expression is driven by endogenous mouseCd276promoter, while mouseCd276gene transcription and translation will be disrupted.

Strain specific MHC I/II expression analysis in wild-type B-NDG mice and homozygous B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice by flow cytometry.Splenocytes were collected from wild-type B-NDG mice (+/+) and homozygous B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice (Mut/Mut, H/H, H/H). Protein expression was analyzed with MHC I antibody (Biolegend, 114613) and MHC II antibody (Biolegend, 109908) by flow cytometry. MHC I and II were only detectable in wild-type B-NDG mice, but not in homozygous B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice.

Western blot analysis of MSLN protein expression in homozygous B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice. Various tissue lysates were collected from wild-type B-NDG mice (+/+) and homozygous B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice (Mut/Mut, H/H, H/H), and then analyzed by western blot with anti-mMSLN antibody (Abcam, ab187063) and anti-hMSLN antibody (Abcam, ab93620). 40 μg total proteins were loaded for western blotting analysis. mMSLN was detected in lung and ovary in wild-type mice, hMSLN was detected in lung and ovary in homozygous B-NDG MHC I/II DKO/hMSLN/hB7-H3 mice.