Basic Information

Strain Name
C57BL/6-Sirpatm1(SIRPA)Cd47tm1(CD47)/Bcgen
Stock Number
120525
Common Name
B-hSIRPA/hCD47 mice
Source/Investigator
Bcgen (Beijing Biocytogen Co., Ltd)
Related Genes
CD47 (CD47 molecule); SIRPα(Signal regulatory protein alpha)
Species
C57BL/6
Appearance
Black
Genotypes
Homozygous
NCBI Gene ID

Targeting Strategy

Gene targeting strategy for B-hSIRPA/hCD47 mice.

The exon 2 of mouse Cd47 gene that encodes the IgV domain was replaced by exon 2 of human CD47 gene.

The exon 2 of mouse Sirpa gene that encodes the IgV domain was replaced by exon 2 of human SIRPA gene in the B-hSIRPA/hCD47 mice.

This double knock-in model was developed by mating the B-hSIRPA mice and the B-hCD47 mice together.

Protein expression analysis

Strain specific CD47 and SIRPα expression analysis in homozygous B-hSIRPA/hCD47 mice by flow cytometry. Splenocytes were collected from WT (+/+) and homozygous B-hSIRPA/hCD47 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-CD47 and anti-SIRPα antibodies. Mouse CD47 was exclusively detectable in WT mice. Mouse SIRPα was detectable in WT mice. This anti-mouse SIRPα antibody also reacts crossly with human SIRPα. Human CD47 and SIRPα were exclusively detectable in homozygous B-hSIRPA/hCD47 mice but not in WT mice.

Peritoneal lymphocytes in B-hSIRPA/hCD47 mice bind to anti-human SIRPα antibody

Analysis of peritoneal lymphocytes of B-hSIRPA/hCD47 mice by flow cytometry. T cells were isolated from female B-hSIRPA/hCD47 mice (n=2, 8-week-old). Flow cytometry analysis of the peritoneal lymphocytes was performed to assess expression of human SIRPα. Single live cells were gated for CD45 population and used for further analysis as indicated here. Human SIRPα was detectable on peritoneal lymphocytes of B-hSIRPA/hCD47 mice as evidenced by anti-human SIRPα antibody binding vs isotype control.

Analysis of spleen leukocyte subpopulations in B-hSIRPA/hCD47 mice

Analysis of spleen leukocyte subpopulations by FACS

Splenocytes were isolated from female C57BL/6 and B-hSIRPA/hCD47 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 CD45 population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, monocytes, DCs, granulocytes and macrophages in homozygous B-hSIRPA/hCD47 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hSIRPα and hCD47 in place of their mouse counterparts does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Blood chemistry of B-hSIRPA/hCD47 mice

Blood chemistry test of B-hSIRPA/hCD47 mice. Plasma from the C57BL/6 and B-hSIRPA/hCD47 mice (n=3, 6 week-old) was collected and analyzed for levels of ALT and AST. There was no differences on either measurement between C57BL/6 and B-hSIRPA/hCD47 mice, indicating that introduction of hSIRPα and hCD47 in place of their mouse counterparts does not change ALT and AST levels or health of liver. Values are expressed as mean ± SEM.

Comparison of hCD47 expression level on red cells between B-hSIRPA/hCD47 mice and human blood

Analysis of hCD47 expression level on red cells in B-hSIRPA/hCD47 mice and human blood by flow cytometry. Red cells were collected from blood of B-hSIRPA/hCD47 mice and human (n=4). The results showed that there was no significant difference in the expression level of human CD47 between B-hSIRPA/hCD47 mice and human red blood cells.

In vivo efficacy of anti-human CD47 antibodies

Antitumor activity of anti-human CD47 antibody in B-hSIRPA/hCD47 mice. (A) anti-human CD47 antibody inhibited MC38-hCD47 tumor growth in B-hSIRPA/hCD47 mice. Murine colon cancer MC38-hCD47 cells were subcutaneously implanted into homozygous B-hSIRPA/hCD47 mice (female, 6-8 week-old, n=5). Mice were grouped when tumor volume reached approximately 150 mm3, at which time they were treated with anti-human CD47 antibody with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, four anti-human CD47 antibodies differently inhibited tumor growth in B-hSIRPA/hCD47 mice, demonstrating that the B-hSIRPA/hCD47 mice provide a powerful preclinical model for in vivo evaluation of anti-human CD47 antibodies. Values are expressed as mean ± SEM.

In vivo efficacy of anti-human SIRPα antibodies

Antitumor activity of anti-human SIRPα antibody in B-hSIRPA/hCD47 mice. (A) anti-human SIRPα antibody inhibited MC38-hCD47 tumor growth in B-hSIRPA/hCD47 mice. Murine colon cancer MC38-hCD47 cells were subcutaneously implanted into homozygous B-hSIRPA/hCD47 mice (female, 6-8 week-old, n=5). Mice were grouped when tumor volume reached approximately 150 mm3, at which time they were treated with anti-human SIRPα antibody with doses and schedules indicated in panel. (B) Body weight changes during treatment. As shown in panel A, three human SIRPA antibodies differently inhibited tumor growth in B-hSIRPA/hCD47 mice, demonstrating that the B-hSIRPA/hCD47 mice provide a powerful preclinical model for in vivo evaluation of anti-human SIRPα antibodies. Values are expressed as mean ± SEM.

Publications using B-hSIRPα/hCD47 mice

  1. Xu Z, Gao J, Yao J, Yang T, Wang D, Dai C, Ding Y. Preclinical efficacy and toxicity studies of a highly specific chimeric anti-CD47 antibody. FEBS Open Bio. 2021 Jan 15. doi: 10.1002/2211-5463.13084. Epub ahead of print. PMID: 33449453.

References

  1. PNAS July 2, 2013 110 (27) 11103-11108; doi: 10.1073/pnas.1305569110
  2. Curr Opin Immunol. 2012 April ; 24(2): 225–232. doi:10.1016/j.coi.2012.01.010
  3.  Genes to Cells (2015) 20, 451–463 doi:10.1111/gtc.12238
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