B-hCD39 mice

Basic Information

Gene Targeting Strategy

Gene targeting strategy for B-hCD39 mice. The coding region sequence that encodes the extracellular domain was inserted followed mouse Entpd1 exon 2 in B-hCD39 mice.

mRNA Expression Analysis

Strain specific analysis of CD39 gene expression in wild-type C57BL/6 mice and B-hCD39 mice by RT-PCR. Mouse Cd39 mRNA was detectable only in splenocytes of wild-type C57BL/6 mice (+/+). Human CD39 mRNA was detectable only in homozygous B-hCD39 mice (H/H), but not in wild-type mice.

Protein Expression Analysis

Strain specific CD39 expression analysis in homozygous B-hCD39 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hCD39 mice, and analyzed by flow cytometry with species-specific CD39 antibody. Mouse CD39 was detectable in WT mice. Human CD39 was exclusively detectable in homozygous B-hCD39 mice but not WT mice.

Analysis of Leukocyte Subpopulations in B-hCD39 Mice

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hCD39 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. (B) Results of FACS analysis. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hCD39 mice were similar to those in the C57BL/6 mice, demonstrating that hCD39 humanized does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Analysis of Spleen T Cell Subpopulations in B-hCD39 Mice

Analysis of spleen T cell subpopulations in B-hCD39 mice. Splenocytes were isolated from female C57BL/6 and B-hCD39 mice (n=3, 6-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live CD45+ cells were gated for CD3+ T cell population and used for further analysis as indicated here. (B) Results of FACS analysis. The percent of Tregs in homozygous B-hCD39 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD39 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes in spleen. Values are expressed as mean ± SEM.

Analysis of Lymph Node Leukocyte Subpopulations in B-hCD39 Mice

Analysis of lymph node leukocyte subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hCD39 mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live cells were gated for CD45+ population and used for further analysis as indicated here. (B) Results of FACS analysis Percent of T cells, B cells in homozygous B-hCD39 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD39 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.

Analysis of Lymph Node Leukocyte Subpopulations in B-hCD39 Mice

Analysis of lymph node leukocyte subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hCD39 mice (n=3, 6- week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live CD45+ cells were gated for CD3+ T cell population and used for further analysis as indicated here. (B) Results of FACS analysis. The percent of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hCD39 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD39 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in lymph node. Values are expressed as mean ± SEM.

Analysis of Blood Leukocyte Subpopulations in B-hCD39 Mice

Analysis of blood leukocyte subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hCD39 mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live cells were gated for CD45+ population and used for further analysis as indicated here. (B) Results of FACS analysis Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hCD39 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD39 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.

Analysis of Blood Leukocyte Subpopulations in B-hCD39 Mice

Analysis of blood leukocyte subpopulations by FACS. Leukocytes were isolated from female C57BL/6 and B-hCD39 mice (n=3, 6-week-old). Flow cytometry analysis of the leukocytes was performed to assess leukocyte subpopulations. (A) Representative FACS plots. Single live CD45+ cells were gated for CD3+ T cell population and used for further analysis as indicated here. (B) Results of FACS analysis. The percent of CD8+ T cells, CD4+ T cells, and Tregs in homozygous B-hCD39 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD39 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in blood. Values are expressed as mean ± SEM.

In Vivo Efficacy of Anti-Human CD39 Antibody

Antitumor activity of anti-human CD39 antibody in B-hCD39 mice. (A) Anti-human CD39 antibody get from cooperation company inhibited MC38 tumor growth in B-hCD39 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hCD39 mice (n=6). Mice were grouped when tumor volume reached approximately 190 mm³, at which time they were treated with anti-human CD39 antibody. (B) Body weight changes during treatment. As shown in panel A, anti-human CD39 antibody were efficacious in controlling tumor growth in B-hCD39 mice with dose dependence. B-hCD39 mice provide a powerful preclinical model for in vivo evaluation of anti-human CD39 antibody. Values are expressed as mean ± SEM.

Summary

mRNA expression analysis:
Mouse CD39 mRNA was detectable only in splenocytes of wild-type C57BL/6 mice (+/+). Human CD39 mRNA was detectable only in homozygous B-hCD39 mice (H/H), but not in wild-type mice.

Protein expression analysis:
Human CD39 was detectable on T cells and B cells in splenocytes of homozygous B-hCD39 mice but not in wild-type mice, and mouse CD39 was detectable only in wild-type mice.

Leukocytes cell subpopulation analysis：
CD39 humanized does not change the overall development, differentiation or distribution of immune cell types in spleen, thymus and blood.

In vivo efficacy：
Anti-human CD39 antibody was effective in controlling tumor growth in B-hCD39 mice.