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B-hSIRPA/hCD47, Prkdc KO mice
Strain Name C57BL/6-Sirpatm1(SIRPA)Cd47tm1(CD47)Prkdctm1/Bcgen Common Name  B-hSIRPA/hCD47, Prkdc KO mice
Background C57BL/6 Catalog number  130565
Aliases 
BIT, CD172A, MFR, MYD-1, P84, PTPNS1, SHPS1, SIRP, IAP, MER6, OA3, DNA-PKC, DNA-PKcs, DNAPK, DNAPKc, DNPK1, HYRC, HYRC1, IMD26, XRCC7, p350, CD132, CIDX

Gene Description


Signal regulatory protein α (SIRPα) is a transmembrane protein with an extracellular region comprising three Ig-like domains and a cytoplasmic region containing immunoreceptor tyrosine-based inhibition motifs which mediate binding of the protein tyrosine phosphatases SHP1 and SHP2. SIRPα is especially abundant in myeloid cells such as macrophages and dendritic cells(DC), whereas it is expressed at very low levels in T, B ,NK, and NK T cells. SIRPα inhibits phagocytosis in macrophages upon interacting with its ligand CD47, which is commonly upregulated on the surface of malignant cells. Thus, antibodies that block the CD47-SIRPα interaction should enhance macrophage phagocytosis in the tumor microenvironment and inhibit tumor growth, making anti-SIRPα antibodies promising tools for cancer immunotherapy. 
Biocytogen has developed the B-hSIRPA/hCD47,Prkdc KO mice. Prkdc gene was knocked out, while the mouse extracellular domain of Sirpa and CD47 genes were replaced with the extracellular domain of human SIRPα and CD47 genes. These mice completely lack T cells and B cells, but express the extracellular domain of human SIRPα and CD47.


Protein expression analysis


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Species specific SIRPα expression analysis in B-hSIRPA/hCD47,Prkdc KO mice by flow cytometry. Splenocytes isolated from C57BL/6 (+/+) mice and homozygous B-hSIRPA/hCD47,Prkdc KO mice (H/H) stimulated with or without anti-mCD3ε in vivo and were analyzed by flow cytometry with anti-SIRPα antibodies. Human SIRPα was exclusively detectable in homozygous B-hSIRPA/hCD47,Prkdc KO mice but not in C57BL/6 mice. Mouse SIRPα was detectable in C57BL/6 and homozygous B-hSIRPA/hCD47,Prkdc KO mice, indicating that this anti-mouse SIRPα antibody was cross-reacting with human SIRPα. 

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Species specific CD47 expression analysis in B-hSIRPA/hCD47,Prkdc KO mice by flow cytometry. Splenocytes were isolated from C57BL/6 (+/+) mice and homozygous B-hSIRPA/hCD47,Prkdc KO mice (H/H) stimulated with or without anti-mCD3ε in vivo and were analyzed by flow cytometry with anti-CD47 antibodies. Mouse CD47 was only detectable in C57BL/6 mice. Human CD47 was exclusively detectable in homozygous B-hSIRPA/hCD47,Prkdc KO mice but not in C57BL/6 mice. Human CD47 was not detectable on T cells in B-hSIRPA/hCD47,Prkdc KO mice stimulated with or without anti-mCD3ε antibody.

Analysis of leukocyte subpopulations in spleen

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Analysis of leukocyte subpopulations in spleen by FACS
Splenocytes were isolated from C57BL/6 and B-hSIRPA/hCD47,Prkdc KO mice (n=2, 5-week-old). Flow cytometry analysis of the splenocytes was performed to assess T cells and B cells. T, B cells were undetectable in B-hSIRPA/hCD47,Prkdc KO mice stimulated with or without anti-mCD3ε antibody.

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Analysis of spleen leukocyte subpopulations by FACS
Splenocytes were isolated from female C57BL/6 and B-hSIRPA/hCD47,Prkdc KO mice (n=3, 8-week-old). Flow cytometry analysis of the splenocytes 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 NK cells, granulocytes, macrophages and monocytes in homozygous B-hSIRPA/hCD47,Prkdc KO mice were increased compared to those in the C57BL/6 mice, while B cells and all T cells were undetectable in B-hSIRPA/hCD47,Prkdc KO mice. Results indicated that knockout of Prkdc gene prevents the development of B cells and T cells in spleen. 

Analysis of T cell subpopulations in spleen

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Analysis of leukocyte subpopulations in spleen by FACS
Splenocytes were isolated from C57BL/6 and B-hSIRPA/hCD47,Prkdc KO mice (n=3, 8-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. CD4+ T cells, CD8+ T cells and Tregs were undetectable in homozygous B-hSIRPA/hCD47,Prkdc KO. Results indicated that knockout of Prkdc gene prevented differentiation of T cells in spleen. 

Analysis of T cell subpopulations in thymus

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Analysis of leukocyte subpopulations in spleen by FACS
Thymocytes were isolated from C57BL/6 and B-hSIRPA/hCD47,Prkdc KO mice (n=3, 8-week-old). Flow cytometry analysis of the thymocytes 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. All subtypes of T cells (CD4-CD8-DN cells, CD4+, CD8+, CD4+CD8+DP T cells and Tregs) were undetectable in thymus.