C57BL/6-Tnftm1(TNF)Bcgen Tnfrsf1btm1(TNFRSF1B)Bcgen Tnfrsf1atm1(TNFRSF1A)Bcgen/Bcgen • 131706
Key Advantages
Validation
Application
In B-hTNFA/hTNFR2/hTNFR1 mice, the targeted mouse Tnfα whole genomic sequence, including 5' UTR, 3' UTR, and coding region, was replaced by the corresponding human TNFα whole genomic sequence.
For receptor humanization, mouse Tnfr2 exons 2-6 encoding the extracellular domain were replaced with human TNFR2 exons 2-6, and mouse Tnfr1 exons 2-6 encoding the extracellular domain were replaced with human TNFR1 exons 2-6. This targeting strategy enables B-hTNFA/hTNFR2/hTNFR1 mice to express human TNFα, humanized TNFR2, and humanized TNFR1 for translational TNF pathway studies.
Strain-specific TNFA protein expression was analyzed in wild-type mice and B-hTNFA/hTNFR2/hTNFR1 mice by ELISA.
Serum was collected from wild-type mice (+/+) and homozygous B-hTNFA/hTNFR2/hTNFR1 mice (H/H;H/H;H/H) after in vivo LPS stimulation and analyzed with species-specific TNFα ELISA kits. Mouse TNFα was detectable in wild-type mice, while human TNFα was detectable in homozygous B-hTNFA/hTNFR2/hTNFR1 mice but not in wild-type controls. Values are expressed as mean ± SEM. ND: not detectable.
Protein Expression Analysis of TNFR1 in B-hTNFA/hTNFR2/hTNFR1 Mice
Strain-specific TNFR1 protein expression was analyzed in homozygous B-hTNFA/hTNFR2/hTNFR1 mice by flow cytometry.
Splenocytes were collected from wild-type mice (+/+) and homozygous B-hTNFA /hTNFR2/hTNFR1 mice (H/H;H/H;H/H), then stained with species-specific anti-TNFR1 antibodies. Mouse TNFR1 was detectable in wild-type mice. Human TNFR1 was detectable in homozygous B-hTNFA/hTNFR2/hTNFR1 mice but not in wild-type controls.
Protein Expression Analysis of TNFR2 in B-hTNFA/hTNFR2/hTNFR1 Mice
Strain-specific TNFR2 protein expression was analyzed in homozygous B-hTNFA/hTNFR2/hTNFR1 mice by flow cytometry.
Splenocytes were collected from wild-type mice (+/+) and homozygous B-hTNFA/hTNFR2/hTNFR1 mice (H/H;H/H;H/H), stimulated with anti-CD3ε in vivo, and stained with species-specific anti-TNFR2 antibodies. Mouse TNFR2 was detectable in wild-type mice. Human TNFR2 was detectable in homozygous B-hTNFA/hTNFR2/hTNFR1 mice but not in wild-type controls.
Spleen leukocyte subpopulations were analyzed by flow cytometry in B-hTNFA/hTNFR2/hTNFR1 mice.
Splenocytes were isolated from wild-type C57BL/6N mice and homozygous B-hTNFA/hTNFR2/hTNFR1 mice (female, 7-week-old, n=3). Flow cytometry was used to evalsuate T cells, B cells, NK cells, dendritic cells, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells, and Tregs. The frequencies of these leukocyte subpopulations in B-hTNFA/hTNFR2/hTNFR1 mice were similar to those in C57BL/6N mice, indicating that TNFα, TNFR2, and TNFR1 humanization does not alter spleen immune-cell distribution. Values are expressed as mean ± SEM.
Blood leukocyte subpopulations were analyzed by flow cytometry in B-hTNFA/hTNFR2/hTNFR1 mice.
Blood cells were isolated from wild-type C57BL/6N mice and homozygous B-hTNFA/hTNFR2/hTNFR1 mice (female, 7-week-old, n=3). Flow cytometry analysis showed that T cells, B cells, NK cells, dendritic cells, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells, and Tregs were similar between B-hTNFA/hTNFR2/hTNFR1 mice and C57BL/6N controls. These results support baseline immune comparability after TNFα/TNFR2/TNFR1 humanization. Values are expressed as mean ± SEM.
Complete blood count (CBC) was performed to evalsuate hematological characteristics of B-hTNFA/hTNFR2/hTNFR1 mice.
Blood samples were collected from wild-type C57BL/6N mice and homozygous B-hTNFA/hTNFR2/hTNFR1 mice for hematology analysis. The measured hematology parameters were comparable between B-hTNFA/hTNFR2/hTNFR1 mice and wild-type controls, supporting normal baseline blood-cell profiles after TNFα, TNFR2, and TNFR1 humanization.
Serum biochemistry analysis was performed to characterize baseline clinical chemistry in B-hTNFA/hTNFR2/hTNFR1 mice.
Serum samples were collected from wild-type C57BL/6N mice and homozygous B-hTNFA/hTNFR2/hTNFR1 mice. Blood chemistry parameters were comparable between B-hTNFA/hTNFR2/hTNFR1 mice and wild-type controls, indicating that TNFα/TNFR2/TNFR1 humanization does not cause overt baseline serum biochemistry abnormalities.
Adalimumab activity was evalsuated in an LPS-induced inflammatory response model using B-hTNFA/hTNFR2/hTNFR1 mice.
Homozygous B-hTNFA/hTNFR2/hTNFR1 mice received PBS or adalimumab (i.v., in-house) two hours before LPS injection (i.p.). Serum was collected one hour and four hours after LPS injection, and human TNFα, mouse IL6, and mouse IFNλ were measured by ELISA. LPS significantly increased human TNFα at one and four hours, mouse IL6 at one and four hours, and mouse IFNλ at four hours. Adalimumab significantly decreased human TNFα at one hour and reduced downstream mouse IL6 and mouse IFNλ at four hours, indicating inhibition of hTNFα-driven activation. Values are expressed as mean ± SEM. Significance was determined by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001.
Anti-human TNFR2 antibody efficacy was evalsuated in MC38 tumor-bearing B-hTNFA/hTNFR2/hTNFR1 mice.
Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hTNFA/hTNFR2/hTNFR1 mice (female, 8-week-old, n=6). Mice were grouped according to body weight differences and treated with anti-TNFR2 Ab1 provided by a client according to the indicated doses and schedules. Anti-human TNFR2 antibodies inhibited MC38 tumor growth in a dose-dependent manner, while body weight was monitored during treatment. These data support B-hTNFA/hTNFR2/hTNFR1 mice as a preclinical model for in vivo evalsuation of anti-human TNFR2 antibodies. Values are expressed as mean ± SEM.
Establishment of CIA Mouse Model
A collagen-induced arthritis (CIA) mouse model was established in B-hTNFA/hTNFR2/hTNFR1 mice.
B-hTNFA/hTNFR2/hTNFR1 mice (10-week-old, 6 males and 10 females) and C57BL/6 mice (10-week-old) were used for CIA model development. Collagen type II (CII) emulsion was used as the modeling reagent. Mice were sensitized on Day 0 and challenged on Day 21 to induce arthritis, providing a TNFα/TNFR2/TNFR1 humanized inflammatory disease model for rheumatoid arthritis research.
Collagen-induced arthritis was induced in B-hTNFA/hTNFR2/hTNFR1 mice and C57BL/6 mice using CII emulsion.
Mouse body weight change and clinical arthritis score were monitored after CIA induction. The clinical score of B-hTNFA/hTNFR2/hTNFR1 mice was significantly increased, demonstrating successful arthritis model establishment. These data support the use of B-hTNFA/hTNFR2/hTNFR1 mice for TNFα-targeted therapy evalsuation in an autoimmune arthritis setting.
Pathological analysis was performed after CIA model establishment in B-hTNFA/hTNFR2/hTNFR1 mice and C57BL/6 mice.
Joint pathology was evalsuated by pathological scoring and H&E staining. In the CIA model group, subcutaneous mixed inflammatory-cell infiltration, periarticular stenosis, articular cartilage destruction, bone tissue destruction, and other arthritis-associated lesions were observed in all or part of the limb joints. These pathological findings confirmed successful establishment of the CIA arthritis model in B-hTNFA/hTNFR2/hTNFR1 mice.
Adalimumab efficacy was evalsuated in B-hTNFA/hTNFR2/hTNFR1 mice with collagen-induced arthritis.
CIA was induced by subcutaneous injection of CII emulsion on Day 0 and Day 21 in female B-hTNFA/hTNFR2/hTNFR1 mice (n=9-10/group). Mice were grouped when inflammation appeared, defined as clinical score >1 or continuous score =1, and treated intraperitoneally with different doses of anti-human TNFα antibody adalimumab (in-house). Body weight change and clinical score were evalsuated daily. Total clinical score increased in the model groups, confirming arthritis establishment, while adalimumab treatment produced a dose-dependent reduction in clinical score. These results support B-hTNFA/hTNFR2/hTNFR1 mice as a CIA model for in vivo anti-human TNFα antibody evalsuation.
Histopathological examination was performed to evalsuate anti-human TNFα antibody efficacy in the CIA model.
At endpoint, joints from B-hTNFA/hTNFR2/hTNFR1 mice were collected, decalcified, sectioned, and stained with H&E. Inflammatory-cell infiltration, synovial hyperplasia, and bone structure damage were assessed by histopathological scoring. The pathological score of the model group was higher than that of the control group, confirming successful modeling. The pathological score was lower in the adalimumab-treated group than in the model group, indicating therapeutic activity of anti-human TNFα antibody in B-hTNFA/hTNFR2/hTNFR1 CIA mice.
Q1: What are B-hTNFA/hTNFR2/hTNFR1 mice?
B-hTNFA/hTNFR2/hTNFR1 mice are triple gene-humanized mice expressing human TNFα, humanized TNFR2, and humanized TNFR1 in a C57BL/6 background for TNF pathway drug development.
Q2: Why are TNFα, TNFR1, and TNFR2 important therapeutic targets?
TNFα signaling through TNFR1 and TNFR2 regulates inflammation, immune activation, autoimmunity, tissue injury, and tumor immunity, making this pathway important for inflammatory disease and oncology therapeutics.
Q3: How were TNFα, TNFR1, and TNFR2 validated in this model?
Human TNFα was validated by ELISA after LPS stimulation, while human TNFR1 and human TNFR2 were validated by flow cytometry using species-specific antibodies.
Q4: Can B-hTNFA/hTNFR2/hTNFR1 mice be used for in vivo antibody efficacy studies?
Yes. The model supports adalimumab evalsuation in LPS-induced response and CIA arthritis models, and anti-human TNFR2 antibody evalsuation in MC38 tumor-bearing mice.
Q5: What are the main applications of B-hTNFA/hTNFR2/hTNFR1 mice?
Applications include anti-human TNFα antibody studies, anti-human TNFR2 antibody research, TNFR1/TNFR2 pathway pharmacology, LPS inflammation models, MC38 tumor studies, and collagen-induced arthritis drug development.
