• TNF encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. This cytokine is mainly secreted by macrophages. It can bind to its receptors TNFRSF1A and TNFRSF1B and exert its effects through these receptors.
• The IL-7 receptor alpha chain (IL-7Ra) and the common gamma chain (γc) serve as the components of the IL-7 receptor, undergoing dimerization upon binding with the IL-7 ligand. IL-7 and its receptor IL-7R are crucial for the normal development, differentiation, and survival of T cells and B cells. Abnormal IL-7/IL-7R signaling is believed to be associated with the pathogenesis of various autoimmune or inflammatory diseases.
• The genome of the mouse Tnf gene encoding the full-length protein was replaced with human TNF counterpart in B-hTNFA/hTNFR2/hTNFR1/hIL7R mice. The genome of the mouse Tnfr1 gene encoding the extracellular domain was replaced with its human TNFR1 counterpart in in B-hTNFA/hTNFR2/hTNFR1/hIL7R mice. The genome of the mouse Tnfr2 gene encoding the extracellular domain was replaced with its human TNFR2 counterpart in in B-hTNFA/hTNFR2/hTNFR1/hIL7R mice. The genome of the mouse Il7r gene encoding the extracellular domain was replaced with human IL7R counterpart in B-hTNFA/hTNFR2/hTNFR1/hIL7R mice.
• Mouse TNFA, TNFR1, TNFR2, and IL7R protein were only detectable in wild-type C57BL/6JNifdc mice, and human TNFA, TNFR1, TNFR2, and IL7R protein were exclusively detectable in homozygous B-hTNFA/hTNFR1/hTNFR2/hIL7R mice.
• Application: This product is used for pharmacodynamics and safety evaluation of drugs.

Gene targeting strategy for B-hTNFA/hTNFR2/hTNFR1/hIL7R mice.

The exons 1-4 of mouse Tnfa gene that encode signal peptide, extracellular domain, transmembrane domain, cytoplasmic region and 3’UTR are replaced by human counterparts in B-hTNFA/hTNFR2/hTNFR1/hIL7R mice. The promoter and 5’UTR region of the mouse gene are replaced by human counterparts. The human TNFA expression is driven by human TNFA promoter, while mouse Tnfa gene transcription and translation will be disrupted.

The exons 2-6 of mouse Tnfr1 gene that encode extracellular domain are replaced by human counterparts in B-hTNFA/hTNFR2/hTNFR1/hIL7R mice. The genomic region of mouse Tnfr1 gene that encodes transmembrane domain and cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric TNFR1 expression is driven by endogenous mouse Tnfr1 promoter, while mouse Tnfr1 gene transcription and translation will be disrupted.

The exons 2-6 of mouse Tnfr2 gene that encode extracellular domain are replaced by human counterparts in B-hTNFA/hTNFR2/hTNFR1/hIL7R mice. The genomic region of mouse Tnfr2 gene that encodes transmembrane domain and cytoplasmic portion is retained. The promoter, 5’UTR and 3’UTR region of the mouse gene are also retained. The chimeric TNFR2 expression is driven by endogenous mouse Tnfr2 promoter, while mouse Tnfr2 gene transcription and translation will be disrupted.

The exons 1-6 of mouse Il7r gene that encode the extracellular region were replaced by human IL7R exons 1-6 in B-hTNFA/hTNFR2/hTNFR1/hIL7R mice.

Strain specific TNFR1 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hTNFA/hTNFR2/hTNFR1/hIL7R mice by flow cytometry.  Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTNFA/hTNFR2/hTNFR1/hIL7R mice(H/H;H/H;H/H;H/H), protein expression was analyzed with anti-mouse TNFR1 antibody (Biolegend, 113005) and anti-human TNFR1 antibody (Biolegend, 369903) by flow cytometry. Mouse TNFR1 was detectable in wild-type C57BL/6JNifdc mice. Human TNFR1 was detectable in homozygous B-hTNFA/hTNFR2/hTNFR1/hIL7R mice. WT: wild-type C57BL/6JNifdc mice; HO: homozygous B-hTNFA/hTNFR2/hTNFR1/hIL7R mice.

Strain specific TNFR2 expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hTNFA/hTNFR2/hTNFR1/hIL7R mice by flow cytometry.  Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTNFA/hTNFR2/hTNFR1/hIL7R mice(H/H;H/H;H/H;H/H), stimulated with anti-CD3ε in vivo (7.5 μg/mice, stimulation for 24 h, i.p.). Protein expression was analyzed with anti-mouse TNFR2 antibody (Biolegend, 113405) and anti-human TNFR2 antibody (Biolegend, 358406)  by flow cytometry. Mouse TNFR2 was detectable in wild-type C57BL/6JNifdc mice. Human TNFR2 was detectable in homozygous B-hTNFA/hTNFR2/hTNFR1/hIL7R mice. WT: wild-type C57BL/6JNifdc mice; HO: homozygous B-hTNFA/hTNFR2/hTNFR1/hIL7R mice.

Strain specific IL7R expression analysis in wild-type C57BL/6JNifdc mice and homozygous B-hTNFA/hTNFR2/hTNFR1/hIL7R mice by flow cytometry.  Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTNFA/hTNFR2/hTNFR1/hIL7R mice(H/H;H/H;H/H;H/H), protein expression was analyzed with anti-mouse IL7R antibody (Biolegend, 135011) and anti-human IL7R antibody (Biolegend, 351303) by flow cytometry. Mouse IL7R was detectable in wild-type C57BL/6JNifdc mice. Human IL7R was detectable in homozygous B-hTNFA/hTNFR2/hTNFR1/hIL7R mice. WT: wild-type C57BL/6JNifdc mice; HO: homozygous B-hTNFA/hTNFR2/hTNFR1/hIL7R mice.

Strain specific TNFA expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hTNFA/hTNFR1/hTNFR2/hIL7R mice by ELISA. Serum were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hTNFA/hTNFR1/hTNFR2/hIL7R mice (H/H;H/H;H/H;H/H), the production of TNFA in serum were assessed after 2 h of stimulation with LPS in vivo. Expression levels of mouse and human TNFA were analyzed by ELISA (Mouse TNFA ELISA Kit: Biolegend, 430904; Human TNFA ELISA Kit: Biolegend, 430204). Mouse TNFA was only detectable in wild-type C57BL/6JNifdc mice, and human TNFA was only detectable in homozygous B-hTNFA/hTNFR1/hTNFR2/hIL7R mice. Values are expressed as mean ± SEM.