Basic Information
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Gene targeting strategy
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Gene targeting strategy for B-hLTA/hTNFA/hLTB mice. Exons 2-4 of mouse Lta gene that encodes the full-length protein and 3’UTR were replaced by human LTA exons 2-4 in B-hLTA/hTNFA/hLTB 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 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 mice.
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mRNA expression analysis
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Species-specific LTA, TNFA, and LTB gene expression analysis in wild-type and humanized B-hLTA/hTNFA/hLTB mice by RT-PCR. Mouse Lta, Tnfa and Ltb mRNAs were detected in splenocytes isolated from wild-type C57BL/6 (+/+) mice, while human LTA, TNFα and LTB mRNAs were detected in homozygous B-hLTA/hTNFA/hLTB mice (H/H) mice.
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Protein expression analysis
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Species-specific TNFA expression analysis in wild-type and humanized B-hLTA/hTNFA/hLTB mice. Serum was collected from wild-type C57BL/6 (+/+) and homozygous B-hLTA/hTNFA/hLTB (H/H) mice stimulated with LPS in vivo, and analyzed by ELISA using species-specific TNFα ELISA kits. Mouse TNFα was detected in wild-type mice, while human TNFα was detected in B-hLTA/hTNFA/hLTB mice.
Species-specific LTB expression analysis in wild-type and humanized B-hLTA/hTNFA/hLTB mice. Spleen and thymic tissues were isolated from wild-type C57BL/6 (+/+) and homozygous B-hLTA/hTNFA/hLTB (H/H) mice, and analyzed by western blot using anti-LTB antibodies. LTB protein was detected in both wild-type and B-hLTA/hTNFA/hLTB mice, due to the cross reactive.
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