• 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. CD20 is a marker of B cells, and it begins to express in Pre-B cells and loses expression after differentiating into plasma cells. CD20 is expressed on the surface of normal and about 95% malignant B lymphocytes, but not expressed in hematopoietic stem cells, plasma cells, or other normal tissues. CD20 is regarded as an ideal target for the treatment of B-cell lymphoma and autoimmune diseases. CD79B encodes an Ig-β protein that forms part of the antigenic component in B cells. The CD79A and CD79B proteins together form a heterodimeric complex, and their interaction with immunoglobulin heavy chains is crucial for the expression of B-cell receptor (BCR) on the cell surface and the subsequent initiation of signaling pathways triggered by BCR activation.
• B-hCD3E/hCD20/hCD79B mice were obtained by mating B-hCD3E mice(110008), B-hCD20 mice(111231) and B-hCD79B mice(111096). In this mice, the exons 2-6 of mouse Cd3e gene that encode the extracellular domain were replaced by human CD3E exons 2-7. The exons 1-4 of mouse Cd79b gene that encode the extracellular domain were replaced by human CD79B exons 1-4 in B-hCD3E/hCD20/hCD79B mice.
• Application: This product is used to assess the pharmacodynamics and toxicity of trispecific antibodies, CAR T-cell therapy, or ADC drugs for treating various B-cell lymphomas.

Gene targeting strategy for B-hCD3E/hCD20/hCD79B mice. The exons 2-6 of mouse Cd3e gene that encode the extracellular domain were replaced by human CD3E exons 2-7 in B-hCD3E/hCD20/hCD79B mice. The exons 2-7 of mouse Cd20 gene that encode the full-length protein were replaced by human CD20 exons 2-7 in B-hCD3E/hCD20/hCD79B mice. The exons 1~4 of mouse Cd79b gene that encode the extracellular domain were replaced by human CD79B exons 1~4 in B-hCD3E/hCD20/hCD79B mice.

Species specific analysis of CD3E, CD20 and CD79B gene expression in wild-type C57BL/6 mice and homozygous humanized B-hCD3E/hCD20/hCD79B mice by RT-PCR. Blood was collected from wild-type C57BL/6 mice and homozygous B-hCD3E/hCD20/hCD79B mice. Mouse Cd3e, Cd20 and Cd79b mRNA were detectable only in wild-type C57BL/6 mice. Human CD3E, CD20 and CD79B mRNA were detectable only in homozygous B-hCD3E/hCD20/hCD79B mice, but not in wild-type C57BL/6 mice.

Species specific analysis of CD3E, CD20 and CD79B gene expression in wild-type C57BL/6 mice and homozygous humanized B-hCD3E/hCD20/hCD79B mice by RT-qPCR. Blood was collected from wild-type C57BL/6 mice and homozygous B-hCD3E/hCD20/hCD79B mice (female, 12-15 week-old, n=3). The mRNA expression of human CD3E, CD20 and CD79B in homozygous B-hCD3E/hCD20/hCD79B mice was similar to those in the wild-type C57BL/6 mice, demonstrating that humanization of CD3E, CD20 and CD79B does not change the CD3E, CD20 and CD79B expression. Values are expressed as mean ± SEM. Significance was determined by unpaired t-test.  

Strain specific CD3E and CD20 expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD3E/hCD20/hCD79B mice by flow cytometry. Splenocytes and blood were collected from wild-type C57BL/6 mice and homozygous B-hCD3E/hCD20/hCD79B mice (H/H). Protein expression was analyzed with anti-mouse CD3E antibody (Biolegend, 100330), anti-human CD3E antibody (BD Pharmingen, 562426), anti-mouse CD20 antibody (Biolegend, 152106) and anti-human CD20 antibody (Biolegend, 302304) by flow cytometry. Mouse CD3E and CD20 were only detectable in wild-type C57BL/6 mice. Human CD3E and CD20 were exclusively detectable in homozygous B-hCD3E/hCD20/hCD79B mice, but not in wild-type C57BL/6 mice.

Strain specific CD79B expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD3E/hCD20/hCD79B mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice and homozygous B-hCD3E/hCD20/hCD79B mice. Protein expression was analyzed with anti-mouse CD79B antibody (RD, FAB6814V) and anti-human CD79B antibody (Biolegend, 341406) by flow cytometry. Mouse CD79B was only detectable in wild-type C57BL/6 mice. Human CD79B was exclusively detectable in homozygous B-hCD3E/hCD20/hCD79B mice, but not in wild-type C57BL/6 mice.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (female, 12-15 weeks-old, n=3) and homozygous B-hCD3E/hCD20/hCD79B mice (female, 12-15 weeks-old, n=4). Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. Frequencies of T cells, B cells, NK cells, DCs, monocytes, macrophages, granulocytes, CD4+ T cells, CD8+ T cells and Tregs in B-hCD3E/hCD20/hCD79B mice were similar to those in C57BL/6 mice, demonstrating that humanization of CD3E, CD20 and CD79B does not change the frequency or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Homozygous CD79B knockout mice exhibit arrested development of B cells at the pro-B cell stage, and the number of B cells in the spleen sharply decreases.. (doi: 10.1084/jem.194.4.455. )

Frequency of leukocyte subpopulations in blood by flow cytometry. Blood cells were isolated from wild-type C57BL/6 mice (female, 12-15 weeks-old, n=3) and homozygous B-hCD3E/hCD20/hCD79B mice (female, 12-15 weeks-old, n=4). Flow cytometry analysis of the blood cells was performed to assess the frequency of leukocyte subpopulations. Frequencies of T cells, B cells, NK cells, DCs, monocytes, macrophages, granulocytes, CD4+ T cells, CD8+ T cells and Tregs in B-hCD3E/hCD20/hCD79B mice were similar to those in C57BL/6 mice, demonstrating that humanization of CD3E, CD20 and CD79B does not change the frequency or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Homozygous CD79B knockout mice exhibit arrested development of B cells at the pro-B cell stage, and the number of B cells in the spleen sharply decreases.. (doi: 10.1084/jem.194.4.455. )

Frequency of leukocyte subpopulations in lymph nodes by flow cytometry. Leukocytes were isolated from wild-type C57BL/6 mice (female, 12-15 weeks-old, n=3) and homozygous B-hCD3E/hCD20/hCD79B mice (female, 12-15 weeks-old, n=4). Flow cytometry analysis of the leukocytes was performed to assess the frequency of leukocyte subpopulations. Percentages of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and Tregs in B-hCD3E/hCD20/hCD79B mice were similar to those in C57BL/6 mice, demonstrating that humanization of CD3E, CD20 and CD79B does not change the frequency or distribution of these cell types in lymph nodes. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in bone marrow by flow cytometry. Bone marrow cells were isolated from wild-type C57BL/6 mice (female, 12-15 weeks-old, n=3) and homozygous B-hCD3E/hCD20/hCD79B mice (female, 12-15 weeks-old, n=4). Flow cytometry analysis of the bone marrow cells was performed to assess the frequency of leukocyte subpopulations. Frequencies of T cells, B cells, NK cells, DCs, monocytes, macrophages, granulocytes, CD4+ T cells, CD8+ T cells and Tregs in B-hCD3E/hCD20/hCD79B mice were similar to those in C57BL/6 mice, demonstrating that humanization of CD3E, CD20 and CD79B does not change the frequency or distribution of these cell types in bone marrow. Values are expressed as mean ± SEM.

Homozygous CD79B knockout mice exhibit arrested development of B cells at the pro-B cell stage, and the number of B cells in the spleen sharply decreases.. (doi: 10.1084/jem.194.4.455. )

Growth curve of B-hCD3E/hCD20/hCD79B mice. Eight-week-old mice were grouped (10 males and 10 females, respectively). Body weight was measured on the same day of every week and lasted for 15 weeks.

Complete blood count (CBC) of B-hCD3E/hCD20/hCD79B mice. Values are expressed as mean ± SD.

Biochemical test of B-hCD3E/hCD20/hCD79B mice. Values are expressed as mean ± SD.

Average weight of the main organs of B-hCD3E/hCD20/hCD79B mice. Values are expressed as mean ± SD.

Histopathological analysis of organs in B-hCD3E/hCD20/hCD79B mice. The main organs of B-hCD3E/hCD20/hCD79B mice were isolated at 23 weeks of age and analyzed with H&E staining (male, n=10; female, n=10). Results showed that no obvious abnormalities were found in all of the organs (heart, liver, spleen, lung, kidney, brain, thymus, stomach, ileum, colon, submandibular lymph nodes, mesenteric lymph nodes, bone marrow, testis, uterus and ovary). Scale bar: 200 μm and 400 μm.