Targeting strategy
Gene targeting strategy for B-h4-1BB/hCD27 mice. The exon 2-7 of mouse 4-1bb gene that encodes the extracellular domain was replaced by human 4-1BB counterpart gene in B-h4-1BB/hCD27 mice. The exons 1-5 of mouse Cd27 gene that encode the extracellular domain were replaced by human CD27 exons 1-5 in B-h4-1BB/hCD27 mice.
Protein expression analysis
Strain specific 4-1BB and CD27 expression analysis in homozygous B-h4-1BB/hCD27 mice by flow cytometry. Splenocytes were collected from wild-type mice (+/+) and homozygous B-h4-1BB/hCD27 mice (H/H) stimulated with anti-CD3ε in vivo (7.5 μg/mice for 24 hours, i.p.), and analyzed by flow cytometry with anti-4-1BB antibody and anti-CD27 antibody. Human 4-1BB and CD27 were exclusively detectable in homozygous B-h4-1BB/hCD27 mice (H/H) but not in wild-type mice. Mouse 4-1BB were detectable in wild-type mice. Mouse CD27 can detectable both in wild-type mice and B-h4-1BB/hCD27 mice (H/H) for anti-mCD27 antibody can binding with human and mouse CD27.
