• 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.
• The coding region genome of mouse Cd20 gene was replaced by human CD20 coding region in B-hCD20 mice.
• Human CD20 were only detectable in homozygous B-hCD20 mice. Humanization of CD20 does not change the overall frequency or distribution of immune cell types in the spleen, blood, or lymph nodes. Humanized of CD20 does not change the blood cell composition and morphology, ALT, or AST levels or health of liver.
• This product is used for pharmacodynamics and safety evaluation of B-cell lymphoma and autoimmune diseases.

Gene targeting strategy for B-hCD20 mice.
The exons 2-7 of mouse Cd20 gene that encode the whole molecule (ATG to STOP codon) are replaced by human counterparts in B-hCD20 mice. The promoter, 5’UTR, and 3’UTR region of the mouse gene are retained. The human CD20 expression is driven by endogenous mouse Cd20 promoter, while mouse Cd20 gene transcription and translation will be disrupted.

Species specific analysis of CD20 gene expression in wild-type C57BL/6 mice and homozygous humanized B-hCD20 mice by RT-PCR.
Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hCD20 mice (H/H). Mouse CD20 mRNA was detectable only in wild-type C57BL/6 mice. Human CD20 mRNA was detectable only in homozygous B-hCD20 mice, but not in wild-type C57BL/6 mice.

Strain specific CD20 expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hCD20 mice by flow cytometry.
Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hCD20 mice (H/H). Protein expression was analyzed with anti-mouse CD20 antibody (Biolegend, 152107) and anti-human CD20 antibody (Biolegend, 302305) by flow cytometry. Mouse CD20 was only detectable in wild-type C57BL/6 mice. Human CD20 was exclusively detectable in homozygous B-hCD20 mice, but not in wild-type C57BL/6 mice.

Frequency of leukocyte subpopulations in the spleen by flow cytometry.
Splenocytes were isolated from wild-type C57BL/6 mice and homozygous B-hCD20 mice (female, 9-week-old, n=3). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hCD20 mice were similar to those in C57BL/6 mice, demonstrating that the humanization of CD20 does not change the frequency or distribution of these cell types in the spleen. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in the blood by flow cytometry.
Blood cells were isolated from wild-type C57BL/6 mice and homozygous B-hCD20 mice (female, 9-week-old, n=3). A. Flow cytometry analysis of the blood cells was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, DCs, monocytes, macrophages, neutrophils, CD4+ T cells, CD8+ T cells and Tregs in B-hCD20 mice were similar to those in C57BL/6 mice, demonstrating that the humanization of CD20 does not change the frequency or distribution of these cell types in the blood. Values are expressed as mean ± SEM.

Frequency of leukocyte subpopulations in the lymph node by flow cytometry.
Leukocytes were isolated from wild-type C57BL/6 mice and homozygous B-hCD20 mice (female, 9-week-old, n=3). A. Flow cytometry analysis of the leukocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequencies of T cell subpopulations. Percentages of T cells, B cells, NK cells, CD4+ T cells, CD8+ T cells and Tregs in B-hCD20 mice were similar to those in C57BL/6 mice, demonstrating that the humanization of CD20 does not change the frequency or distribution of these cell types in the lymph node. Values are expressed as mean ± SEM.

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

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

B-hCD20 mice (n=6) were intraperitoneally immunized with OVA/CFA or PBS on Day 0. Blood was collected on Day 0, Day 14 and Day 21 and analyzed by ELISA with mouse IgG antibody and OVA specific IgG antibody (A). Concentration of mouse IgG and mouse OVA specific IgG were significantly increased after immunization. This indicates that humanized CD20 does not affect B cell-related humoral immunity.

Sterile anti–human CD20 antibody Obinutuzumab analog (in house), Rituximab analog (in house) and the control  hIgG1 were administered into B-hCD20 mice (female, 6-week-old, n=3) through a single tail vein injection. Blood were collected after treatment. After red blood cell lysis, the frequency of mCD19+ B cell was analyzed by flow cytometry analysis. (A) The proportion of B cells in the CD45+ cell during treatment. (B) Representative FACS images of B cells in the blood. The results indicate that Obinutuzumab analog (in house)  and Rituximab analog (in house) can effectively eliminate B cells in B-hCD20 mice.