Basic Information
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Gene editing strategy
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Gene targeting strategy for B-Rag2/Il2rg DKO mice.
The exon 3 of mouse Rag2 gene that encodes most of the full-length protein and 3’UTR region were knocked out in B-Rag2 KO mice. The exons 1~8 of mouse Il2rg gene that encode the full-length protein were knocked out in B-Il2rg KO mice. B-Rag2 KO mice and B-Il2rg KO mice were crossed together and get the B-Rag2/Il2rg DKO mice.
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The size and weight of thymus and spleen significantly reduced in B-Rag2/Il2rg DKO mice
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The size and weight of thymus and spleen of B-Rag2/Il2rg DKO mice were significantly reduced compared to that of wild-type C57BL/6 mice. Thymuses and spleens were isolated from wild-type C57BL/6 mice and B-Rag2/Il2rg DKO mice (n=3, 7-week-old). A. Gross anatomy of thymuses; B. The size of spleen and thymus; C. Comparison of organ coefficients of spleen and thymus in wild-type C57BL/6 mice and B-Rag2/Il2rg DKO mice. Results showed that thymuses were not visualized in B-Rag2/Il2rg DKO mice. The size and weight of spleens in B-Rag2/Il2rg DKO mice were significantly reduced compared to that in wild-type C57BL/6 mice.
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Evans blue staining of lymph nodes
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Lymph nodes were not visualized in B-Rag2/Il2rg DKO mice dyed by Evans blue.
Wild-type C57BL/6 mice and B-Rag2/Il2rg DKO mice (n=3) were sacrificed and intravenously injected with 10% Evens blue in the planta pedis. The red arrows indicate the lymph nodes in inguinal, axillary and mesenteric site of the mice. No lymph node was visualized in B-Rag2/Il2rg DKO mice.
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Frequency of leukocyte subpopulations in bone marrow
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Frequency of leukocyte subpopulations in bone marrow by flow cytometry. Bone marrow cells were isolated from male wild-type C57BL/6 mice and homozygous B-Rag2/Il2rg DKO mice (n=3, 6-week-old). A. Flow cytometry analysis of the bone marrow cells was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. T cells, including CD4+ T cells, CD8+ T cells and Tregs, B cells and NK cells were not detectable in B-Rag2/Il2rg DKO mice. Frequency of neutrophils, monocytes and macrophages were increased in B-Rag2/Il2rg DKO mice compared to that in C57BL/6 mice, demonstrating that the development of T cells, B cells and NK cells in bone marrow were completely inhibited in B-Rag2/Il2rg DKO mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, **P < 0.01, ***p < 0.001.
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Frequency of leukocyte subpopulations in spleen
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Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from male wild-type C57BL/6 mice and homozygous B-Rag2/Il2rg DKO mice (n=3, 6-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. T cells, including CD4+ T cells, CD8+ T cells and Tregs, B cells and NK cells were not detectable in B-Rag2/Il2rg DKO mice. Frequency of dendritic cells, neutrophils, monocytes and macrophages were increased in B-Rag2/Il2rg DKO mice compared to that in C57BL/6 mice, demonstrating that the development of T cells, B cells and NK cells in spleen were completely inhibited in B-Rag2/Il2rg DKO mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, **P < 0.01, ***p < 0.001.
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Frequency of leukocyte subpopulations in blood
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Frequency of leukocyte subpopulations in blood by flow cytometry. Blood cells were isolated from male wild-type C57BL/6 mice and homozygous B-Rag2/Il2rg DKO mice (n=3, 6-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. T cells, including CD4+ T cells, CD8+ T cells and Tregs, B cells and NK cells were not detectable in B-Rag2/Il2rg DKO mice. Frequency of dendritic cells, neutrophils and macrophages were increased in B-Rag2/Il2rg DKO mice compared to that in C57BL/6 mice, demonstrating that the development of T cells, B cells and NK cells in blood were completely inhibited in B-Rag2/Il2rg DKO mice. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test. *P < 0.05, **P < 0.01, ***p < 0.001.
