• CD3 consists of four protein chains (CD3E, CD3D, CD3G and CD3Z), which are important biological markers on the T cell membrane. CD3 can form a TCR/CD3 complex with the T cell receptor, participating in the regulation of T cell antigen recognition, signal transduction and T cell development. CD28 (Cluster of Differentiation 28) is one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival.
• In B-hCD3EDG/hCD28 mice(C.B6), humanized CD3EDG was expressed, while mouse Cd3edg were knocked out. The exons 2-3 of mouse Cd28 gene that encode the extracellular domain were replaced by human CD28 exons 2-3 in B-hCD3EDG/hCD28 mice(C.B6).
• Human CD3E and CD28 were detectable on T cells of homozygous B-hCD3EDG/hCD28 mice(C.B6) but not in wild-type  BALB/cCrSlcNifdc mice.
• This product is used for the pharmacological and safety evaluation of B-cell lymphoma and autoimmune diseases.

Gene targeting strategy for B-hCD3EDG/hCD28 mice(C.B6). The targeting strategy of humanized CD3EDG were undisclosed. The exons 2-3 of mouse Cd28 gene that encode the extracellular domain were replaced by human CD28 exons 2-3 in B-hCD3EDG/hCD28 mice(C.B6). 

CD3E expression analysis in wild-type mice and homozygous humanized B-hCD3EDG/hCD28 mice(C.B6) by flow cytometry. Spleen T cells and blood T cells were collected from BALB/cCrSlcNifdc mice (+/+) (Female, 6-week-old, n=1) and B-hCD3EDG/hCD28 mice(C.B6) (H/H) (Female, 6-week-old, n=1). Protein expression was analyzed with anti-human CD3E antibody (BD Horizon™, 562426) and anti-mouse CD3E antibody (Biolegend, 100312) by flow cytometry. Human CD3E was exclusively detected in homozygous B-hCD3EDG/hCD28 mice​​(C.B6), confirming successful humanization of CD3E expression.

CD28 expression analysis in wild-type mice and homozygous humanized B-hCD3EDG/hCD28 mice(C.B6) by flow cytometry. Spleen T cells were collected from BALB/cCrSlcNifdc mice and B-hCD3EDG/hCD28 mice(C.B6) (female, 5-week-old; n=1 wild-type +/+, n=2 homozygous H/H). Cells were stimulated in vivo via intraperitoneal injection of 7.5 μg/mouse anti-CD3ε antibodies (anti-mouse CD3ε: clone 145-2C11, BioXCell BE0001-1; anti-human CD3ε: clone OKT3, BioXCell BE0001-2) for 24 hours. Protein expression was analyzed by flow cytometry using species-specific antibodies: anti-mouse CD28 (BioLegend 102105) and anti-human CD28 (BioLegend 302912). ​​Human CD28 was exclusively detected in homozygous B-hCD3E/hCD28 mice​​, confirming successful humanization of CD28 expression.