Lab Anim Res.  2018 Dec;34(4):302-310. 10.5625/lar.2018.34.4.302.

CRISPR/Cas9-mediated knockout of CD47 causes hemolytic anemia with splenomegaly in C57BL/6 mice

  • 1Graduate School of Translational Medicine, Seoul National University College of Medicine, Seoul, Korea.
  • 2Department of Experimental Animal Research, Biomedical Research Institute, Seoul National University Hospital, Seoul, Korea.
  • 3Department of Pathology, Dongguk University Ilsan Hospital, Goyang, Korea.
  • 4College of Medicine Severance Biomedical Science Institute, Yonsei University, Seoul, Korea.
  • 5Department of Biochemistry, Yonsei University, Seoul, Korea.
  • 6Biomedical Center for Animal Resource and Development, Seoul National University, College of Medicine, Seoul, Korea.
  • 7Designed Animal and Transplantation Research Institute, Institute of Green Bio Science Technology, Seoul National University, Pyeongchang-gun, Korea.


CD47 (integrin-associated protein), a multi-spanning transmembrane protein expressed in all cells including red blood cells (RBCs) and leukocytes, interacts with signal regulatory protein α (SIRPα) on macrophages and thereby inhibits phagocytosis of RBCs. Recently, we generated a novel C57BL/6J CD47 knockout (CD47(−/−) hereafter) mouse line by employing a CRISPR/Cas9 system at Center for Mouse Models of Human Disease, and here report their hematological phenotypes. On monitoring their birth and development, CD47(−/−) mice were born viable with a natural male-to-female sex ratio and normally developed from birth through puberty to adulthood without noticeable changes in growth, food/water intake compared to their age and sex-matched wild-type littermates up to 26 weeks. Hematological analysis revealed a mild but significant reduction of RBC counts and hemoglobin in 16 week-old male CD47(−/−) mice which were aggravated at the age of 26 weeks with increased reticulocyte counts and mean corpuscular volume (MCV), suggesting hemolytic anemia. Interestingly, anemia in female CD47(−/−) mice became evident at 26 weeks, but splenomegaly was identified in both genders of CD47(−/−) mice from the age of 16 weeks, consistent with development of hemolytic anemia. Additionally, helper and cytotoxic T cell populations were considerably reduced in the spleen, but not in thymus, of CD47(−/−) mice, suggesting a crucial role of CD47 in proliferation of T cells. Collectively, these findings indicate that our CD47(−/−) mice have progressive hemolytic anemia and splenic depletion of mature T cell populations and therefore may be useful as an in vivo model to study the function of CD47.


CRISPR/Cas9; CD47; hemolytic anemia; splenomegaly

MeSH Terms

Anemia, Hemolytic*
Erythrocyte Indices
Reticulocyte Count
Sex Ratio
Thymus Gland


  • Figure 1 CRISPR/Cas9-mediated gene-targeting strategy to generate CD47−/− mice. (A) A schematic drawing shows the sequence of the CD47 alleles in the wild-type (WT) and CD47−/− (KO) location of the start codon (green), RNA-guided engineered nucleases (RGEN) target sequence (blue) and protospacer adjacent motif (PAM; red). The double-strand break induced by the CRISPR/Cas9 was repaired by non-homologous end joining, resulting in a frameshift mutation by deleting 26 nucleotides and thereby creation of a premature termination codon (asterisk mark). (B) PCR products amplified from the targeted region of genomic DNA revealed genotypes of mice.

  • Figure 2 Body weight change, food and water consumption of CD47−/− mice. (A) The body weight, (B) daily food intake and (C) water consumption of wildtype (WT) and CD47−/− mice were measured weekly for 26 weeks. No significant change was observed in any of the parameters between WT male (open squares) and CD47−/− male (filled squares), and between WT female (open circles) and CD47−/− female (filled circles) mice.

  • Figure 3 Hematological analysis of peripheral blood from CD47−/− mice. (A) 16 week-old of WT and CD47−/− mice (n=3 per group). Significant decrease of RBC counts and hemoglobin was observed in CD47−/− males compared to their wild-type counterparts (black bars for WT and dark gray bars for CD47−/− mice). (B) In the blood from 26 week-old of WT and CD47−/− mice (n=7 per group), RBC counts, hemoglobin and hematocrit were significantly declined in both male and female (light gray bars for WT and white bars for CD47−/− mice), while reticulocyte counts in both genders and mean corpuscular mean volume (MCV) only in male were increased. *P<0.05 and **P<0.01.

  • Figure 4 Increased size and weight of spleens in CD47−/− mice. Representative images and bar graphs show increased size and weight of spleens in the 16 week-old (A) and 26 week-old (B) CD47−/− mice compared to their respective wild-type littermates. The graphs are expressed as a percentage of organ weight/body weight (black bars, WT male; dark gray bars, CD47−/− male; light gray bars, WT female; and white bars, CD47−/− female mice). Scale bar; 5 mm. *P<0.05 and ***P<0.001.

  • Figure 5 Intracellular accumulation of iron-containing hemosiderin in the splenic cells of 26-week-old CD47−/− mice. Representative images of H&E stained spleen tissues from WT (A and C) and CD47−/− mice (B and D) show brown-gold pigmented cells (arrowheads). The pigmented cells were more frequently observed in CD47−/− compared to WT mice. Prussian blue staining (E and F) revealed that there were more iron-accumulated cells (arrows) in the spleens of CD47−/− mice than WT. Scale bars for the images with low magnification (x10) and high magnification (x60) are 250 and 2 µm, respectively.

  • Figure 6 Flow cytometric analysis of cells isolated from the spleens, thymuses and bone marrow of CD47−/− mice. (A and D) Dot plots display CD3+CD5+, CD3+CD4+ and CD3+CD8+ T cell populations as well as CD19+B220+ B cell population in the spleen. All the T cell populations detected here were significantly decreased in CD47−/− mice compared to WT. (B and E) Cells from the bone marrow show no changes in CD19+B220+ B cells between WT and CD47−/− mice. (C and F) The numbers of CD3+CD8+ T cells isolated from thymuses are comparable between WT and CD47−/− mice. **P<0.01, NS: Not significant.


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