C57BL/6-Ltatm1(LTA)Bcgen Tnftm1(TNF)BcgenLtbtm1(LTB)BcgenTnfrsf1btm1(TNFRSF1B)BcgenTnfrsf1atm1(TNFRSF1A)Bcgen Ltbrtm1(LTBR)Bcgen/Bcgen • 113337
| Product name | B-hLTA/hTNFA/hLTB/hTNFR2/hTNFR1/hLTBR mice |
|---|---|
| Catalog number | 113337 |
| Strain name | C57BL/6-Ltatm1(LTA)Bcgen Tnftm1(TNF)BcgenLtbtm1(LTB)BcgenTnfrsf1btm1(TNFRSF1B)BcgenTnfrsf1atm1(TNFRSF1A)Bcgen Ltbrtm1(LTBR)Bcgen/Bcgen |
| Strain background | C57BL/6 |
| NCBI gene ID | 16992, 21926, 16994, 21938, 21937, 17000 |
| Aliases | LT, TNFB, TNFSF1, TNLG1E;DIF-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; |
Description
Targeting strategyGene 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)
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
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
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
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.
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
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
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.
