Gene targeting strategy for B-hCD28/hB7-H3 mice. The exons 2-3 of mouse Cd28 gene that encode the extracellular domain were replaced by human CD28 exons 2-3 in B-hCD28/hB7-H3 mice. The exons 3~4 of mouse Cd276 gene that encode the extracellular domain were replaced by human CD276 exons 3~6 in B-hCD28/hB7-H3 mice. 

Strain specific CD28 expression analysis in homozygous B-hCD28/hB7-H3 mice by flow cytometry. Splenocytes were collected from wild type (WT) mice (+/+) and homozygous B-hCD28/hB7-H3 mice (H/H;H/H), and analyzed by flow cytometry with species-specific anti-CD28 antibody. Mouse CD28 was detectable in WT mice (+/+). Human CD28 was exclusively detectable in homozygous B-hCD28/hB7-H3 mice (H/H;H/H) but not in WT mice (+/+).

Strain specific B7-H3 expression analysis in homozygous B-hCD28/hB7-H3 mice by western blot. Epididymis were collected from wild type (WT) mice (+/+) and homozygous B-hCD28/hB7-H3 mice (H/H;H/H), and analyzed by western blot with anti-B7-H3 antibody. B7-H3 was detectable in WT mice (+/+) and homozygous B-hCD28/hB7-H3 mice (H/H;H/H) due to the cross-reactivity of antibodies.