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B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice
Strain Name

C57BL/6-Ltatm1(LTA)Bcgen Tnftm1(TNF)Bcgen Ltbtm1(LTB)Bcgen Tnfrsf1btm1(TNFRSF1B)Bcgen Tnfrsf1atm1(TNFRSF1A)Bcgen Ltbrtm1(LTBR)Bcgen/Bcgen

Common Name  B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice
Background C57BL/6 Catalog number  113337
Aliases

LT, TNFB, TNFSF1, TNLG1E; DIF, IMD127-alpha, TNFA, TNFSF2, TNLG1F, TNF; TNFC, TNFSF3, TNLG1C, p33; CD120b, TBPII, TNF-R-II, TNF-R75, TNFBR, TNFR1B, TNFR2, TNFR80, p75, p75TNFR; CD120a, FPF, TBP1, TNF-R, TNF-R-I, TNF-R55, TNFAR, TNFR1, TNFR55, TNFR60, p55, p55-R, p60; D12S370, LT-BETA-R, TNF-R-III, TNFCR, TNFR-RP, TNFR2-RP, TNFR3, TNFRSF3


NCBI Gene  ID
4049, 7124, 4050, 7133, 7132, 4055
Description

  • The exons 2-4 of mouse Lta gene that encode the full-length protein and 3’UTR were replaced by human LTA exons 2-4 in B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. The exons 1-4 of mouse Tnf gene including 5'UTR, 3'UTR and coding region were replaced by human TNF exons 1-4 in B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. The exons 1-3 of mouse Ltb gene that encode the full-length protein and 3’UTR were replaced by human LTB exons 1-4 in B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. The exons 2-6 of mouse Tnfr2 gene that  encode the extracellular domain were replaced by human TNFR2 exons 2~6 in B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. The exons 2-6 of mouse Tnfr1 gene that  encode the extracellular domain were replaced by human TNFR1 exons 2-6 in B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. The exons 1-7 of mouse Ltbr gene that encode the extracellular domain were replaced by human LTBR exons 1-7 in B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. 
  • LTA protein was detectable in wild-type C57BL/6 mice and homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice, as the human TNF beta ELISA Kit can cross-recognize both human and mouse LTA. Mouse TNFA protein was only detectable in wild-type C57BL/6 mice, and human TNFA protein was only detectable in homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. LTB protein was detectable in wild type mice and homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice, as the antibody can cross-recognize both human and mouse LTB. Mouse TNFR2 protein was only detectable in wild-type mice. Human TNFR2 protein was only detectable in homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. Mouse TNFR1 was detectable on neutrophils of wild-type mice. Human TNFR1 was detectable on neutrophils of homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. Mouse LTBR protein was detectable in wild-type mice. Human LTBR protein was detectable in homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice.
  • Application: This product is used for pharmacodynamics and safety evaluation of  drugs.

Targeting strategy

Gene targeting strategy for B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice
The exons 2-4 of mouse Lta gene that encode the full-length protein and 3’UTR were replaced by human LTA exons 2-4 in B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. 
The exons 1-4 of mouse Tnf gene including 5'UTR, 3'UTR and coding region were replaced by human TNF exons 1-4 in B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice.
The exons 1-3 of mouse Ltb gene that encode the full-length protein and 3’UTR were replaced by human LTB exons 1-4 in B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. 
The exons 2-6 of mouse Tnfr2 gene that  encode the extracellular domain were replaced by human TNFR2 exons 2-6 in B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. 
The exons 2-6 of mouse Tnfr1 gene that  encode the extracellular domain were replaced by human TNFR1 exons 2-6 in B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. 
The exons 1-7 of mouse Ltbr gene that encode the extracellular domain were replaced by human LTBR exons 1-7 in B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. 

Protein expression analysis in regulatory T cells (Tregs)

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Strain specific TNFR2 expression analysis in wild-type C57BL/6 mice and homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice, stimulated with anti-CD3ε in vivo (7.5 μg/mice, stimulation for 24 hours, i.p.). The protein expression was analyzed with anti-mouse TNFR2 antibody (Biolegend, 113405) and anti-human TNFR2 antibody (Biolegend, 358406) by flow cytometry. Mouse TNFR2 was only detectable in wild-type mice. Human TNFR2 was only detectable in homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice.

Protein expression analysis in neutrophils

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Strain specific TNFR1 expression analysis in wild-type C57BL/6 mice and homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice, 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 mice. Human TNFR1 was detectable in homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice.

Protein expression analysis in macrophages

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Strain specific LTBR expression analysis in wild-type C57BL/6 mice and homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice by flow cytometry. Spleen, blood, and bone marrow were collected from wild-type C57BL/6 mice and homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice, protein expression was analyzed with anti-mouse LTBR antibody (Biolegend, 134407) and anti-human LTBR antibody (Biolegend, 322008) by flow cytometry. Mouse LTBR was detectable in wild-type mice. Human LTBR was detectable in homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice.

Protein expression analysis in serum

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Strain specific LTA expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice by ELISA. Serum were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice (H/H,H/H,H/H,H/H,H/H,H/H), the production of LTA in serum were assessed after 2 hours of stimulation with LPS in vivo. Expression level of human LTA were analyzed by ELISA (Human TNF beta ELISA Kit: Proteintech, KE00386). Human LTA was detectable in wild-type C57BL/6 mice and homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice, as the human TNF beta ELISA Kit can cross-recognize both human and mouse LTA. Values are expressed as mean ± SEM. 

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Strain specific TNFA expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice by ELISA. Serum was collected from wild-type C57BL/6 mice (+/+) and homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice (H/H,H/H,H/H,H/H,H/H,H/H), the production of TNFA in serum were assessed after 2 hours 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/6 mice, and human TNFA was only detectable in homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice. Values are expressed as mean ± SEM. 

Protein expression analysis

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Strain specific LTB expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice by western blot. Spleen was collected from wild-type C57BL/6 mice(+/+) and homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice(H/H,H/H,H/H,H/H,H/H,H/H), stimulated with LPS in vivo for 2 hours, and analyzed by western blot with anti-LTB antibody (Abcam, ab64835). 40 μg total proteins were loaded for western blotting analysis. Mouse LTB was detectable both in wild-type mice and homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice, as the antibody can cross-recognize both human and mouse LTB.

mRNA expression analysis

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Species specific analysis of LTA and LTB gene expression in wild-type C57BL/6 mice and homozygous humanized B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice by RT-PCR. Spleen was collected from wild-type C57BL/6 mice(+/+) and homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice(H/H,H/H,H/H,H/H,H/H,H/H). Mouse Lta and Ltb were detectable in wild-type C57BL/6 mice. Human LTA and LTB mRNA were detectable only in homozygous B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice.