Basic Information
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Targeting strategy
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Gene targeting strategy for B-h4-1BB/hHER2 mice.
The exons 2-7 of mouse Tnfrsf9 gene that encode the extracellular domain were replaced by human TNFRSF9 exons 3-8 in B-h4-1BB/hHER2 mice. The exons 2-17 of mouse Erbb2 gene that encode the extracellular and transmembrane domains were replaced by human ERBB2 exons 2-17 in B-h4-1BB/hHER2 mice.
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mRNA expression analysis
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Strain specific analysis of 4-1BB gene expression in wild type (WT) mice and B-h4-1BB/hHER2 mice by RT-PCR.
Mouse Tnfrsf9 mRNA was detectable only in spleen of WT mice (+/+). Human TNFRSF9 mRNA was detectable only in homozygous B-h4-1BB/hHER2 mice (H/H;H/H) but not in WT mice (+/+).
Strain specific analysis of HER2 gene expression in wild type (WT) mice and B-h4-1BB/hHER2 mice by RT-PCR.
Mouse Erbb2 mRNA was detectable only in liver of WT mice (+/+). Human ERBB2 mRNA was detectable only in homozygous B-h4-1BB/hHER2 mice (H/H;H/H) but not in WT mice (+/+).
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Protein expression analysis
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Strain specific 4-1BB expression analysis in homozygous B-h4-1BB/hHER2 mice by flow cytometry.
Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-h4-1BB/hHER2 mice (H/H;H/H) stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-4-1BB antibody. Mouse 4-1BB was detectable in WT mice (+/+). Human 4-1BB was exclusively detectable in homozygous B-h4-1BB/hHER2 mice (H/H;H/H) but not in WT mice (+/+).
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IHC analysis of HER2 expression
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Immunohistochemical (IHC) analysis of HER2 expression in homozygous B-h4-1BB/hHER2 mice.
The liver and mammary gland were collected from WT mice and homozygous B-h4-1BB/hHER2 mice and analyzed by IHC with anti-HER2 antibody. HER2 was detectable in WT mice and homozygous B-h4-1BB/hHER2 mice due to the cross-reactivity of the antibody. The arrow indicates tissue cells with positive HER2 staining (brown).
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Summary
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mRNA expression analysis:
Mouse Tnfrsf9 mRNA was detectable only in spleen of WT mice (+/+). Human TNFRSF9 mRNA was detectable only in homozygous B-h4-1BB/hHER2 mice (H/H;H/H) but not in WT mice (+/+).
Mouse Erbb2 mRNA was detectable only in liver of WT mice (+/+). Human ERBB2 mRNA was detectable only in homozygous B-h4-1BB/hHER2 mice (H/H;H/H) but not in WT mice (+/+).
Protein expression analysis:
Human 4-1BB was exclusively detectable on T cells of homozygous B-h4-1BB/hHER2 mice (H/H;H/H) but not in WT mice (+/+), and mouse 4-1BB/hHER2 was detectable only in WT mice (+/+).
HER2 was detectable in liver and mammary gland of WT mice (+/+) and homozygous B-h4-1BB/hHER2 mice (H/H;H/H) due to the cross-reactivity of antibodies.