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CRISPR/Cas9-mediated knockout of CD47 causes hemolytic anemia with splenomegaly in C57BL/6 mice
Laboratory Animal Research volume 34, pages302–310(2018)
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.
Lindberg FP, Lublin DM, Telen MJ, Veile RA, Miller YE, Donis-Keller H, Brown EJ. Rh-related antigen CD47 is the signal-transducer integrin-associated protein. J Bio Chem 1994; 269(3): 1567–1570.
Reinhold MI, Lindberg FP, Plas D, Reynolds S, Peters MG, Brown EJ. In vivo expression of alternatively spliced forms of integrin-associated protein (CD47). J Cell Sci 1995; 108(11): 3419–3425.
Lindberg FP, Bullard DC, Caver TE, Gresham HD, Beaudet AL, Brown EJ. Decreased resistance to bacterial infection and granulocyte defects in IAP-deficient mice. Science 1996; 274(5288): 795–798.
Oldenborg PA, Zheleznyak A, Fang Y-F, Lagenaur CF, Gresham HD, Lindberg FP. Role of CD47 as a marker of self on red blood cells. Science 2000; 288(5473): 2051–2054.
Armant M, Avice M-N, Hermann P, Rubio M, Kiniwa M, Delespesse G, Sarfati M. CD47 ligation selectively downregulates human interleukin 12 production. J Exp Med 1999; 190(8): 1175–1182.
Demeure CE, Tanaka H, Mateo V, Rubio M, Delespesse G, Sarfati M. CD47 engagement inhibits cytokine production and maturation of human dendritic cells. J Immunol 2000; 164(4): 2193–2199.
Mateo V, Lagneaux L, Bron D, Biron G, Armant M, Delespesse G, Sarfati M. CD47 ligation induces caspase-independent cell death in chronic lymphocytic leukemia. Nat Med 1999; 5(11): 1277–1284.
Waclavicek M, Majdic O, Stulnig T, Berger M, Baumruker T, Knapp W, Pickl WF. T cell stimulation via CD47: agonistic and antagonistic effects of CD47 monoclonal antibody 1/1A4. J Immunol 1997; 159(11): 5345–5354.
Lavender KJ, Pang WW, Messer RJ, Duley AK, Race B, Phillips K, Scott D, Peterson KE, Chan CK, Dittmer U, Dudek T, Allen TM, Weissman IL, Hasenkrug KJ. BLT-humanized C57BL/6 Rag2-/-?c-/-CD47-/- mice are resistant to GVHD and develop B-and T-cell immunity to HIV infection. Blood 2013; 122(25): 4013–4
Lavender KJ, Messer RJ, Race B, Hasenkrug KJ. Production of bone marrow, liver, thymus (BLT) humanized mice on the C57BL/6 Rag2(-/-)?c(-/-)CD47(-/-) background. J Immunol Methods 2014; 407: 127–134.
Van PUTTEN LM, Croon F. The life span of red cells in the rat and the mouse as determined by labeling with DFP32 in vivo. Blood 1958; 13(8): 789–794.
Oldenborg PA, Gresham HD, Chen Y, Izui S, Lindberg FP. Lethal autoimmune hemolytic anemia in CD47-deficient nonobese diabetic (NOD) mice. Blood 2002; 99(10): 3500–3504.
Lee JH, Park JH, Nam TW, Seo SM, Kim JY, Lee HK, Han JH, Park SY, Choi YK, Lee HW. Differences between immunodeficient mice generated by classical gene targeting and CRISPR/Cas9-mediated gene knockout. Transgen Res 2018; 27(3): 241–251.
Council NR. Guide for the care and use of laboratory animals: National Academies Press; 2010.
Kim JI, Park JS, Kim H, Ryu SK, Kwak J, Kwon E, Yun JW, Nam KT, Lee HW, Kang BC. CRISPR/Cas9-mediated knockout of Rag-2 causes systemic lymphopenia with hypoplastic lymphoid organs in FVB mice. Lab Anim Res 2018; 34(4): 166–175.
Wennberg E, Weiss L. Splenomegaly and hemolytic anemia induced in rats by methylcellulose-an electron microscopic study. J Morphol 1967; 122(1): 35–61.
Wagner KU, Claudio E, Rucker EB 3rd, Riedlinger G, Broussard C, Schwartzberg PL, Siebenlist U, Hennighausen L. Conditional deletion of the Bcl-x gene from erythroid cells results in hemolytic anemia and profound splenomegaly. Development 2000; 127(22): 4949–4958.
Sato-Hashimoto M, Saito Y, Ohnishi H, Iwamura H, Kanazawa Y, Kaneko T, Kusakari S, Kotani T, Mori M, Murata Y, Okazawa H, Ware CF, Oldenborg PA, Nojima Y, Matozaki T. Signal regulatory protein a regulates the homeostasis of T lymphocytes in the spleen. J Immunol 2011; 187(1): 291–297.
Guimont-Desrochers F, Beauchamp C, Chabot-Roy G, Dugas V, Hillhouse EE, Dusseault J, Langlois G, Gautier-Ethier P, Darwiche J, Sarfati M, Lesage S. Absence of CD47 in vivo influences thymic dendritic cell subset proportions but not negative selection of thymocytes. Int Immunol 2009; 21(2): 167–177.
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Kim, J., Park, J., Kwak, J. et al. CRISPR/Cas9-mediated knockout of CD47 causes hemolytic anemia with splenomegaly in C57BL/6 mice. Lab Anim Res 34, 302–310 (2018). https://doi.org/10.5625/lar.2018.34.4.302
- hemolytic anemia