Basic Information
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Gene targeting strategy
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Gene targeting strategy for B-Rag2 KO mice. The exon 3 and 3’UTR regions were knocked out in B-Rag2 KO mice, resulting in a disruption of the Rag2 gene.
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Immune cell analysis
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Analysis of immune cell subpopulations in spleen
Analysis of splenic immune cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-Rag2 KO 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 the CD45+ population and used for further analysis as indicated here. (B) Results of FACS analysis. Compared to cells in wild-type C57BL/6 mice, percent of T cells and B cells were significantly decreased, while percent of all the other cells, including NK cells, dendritic cells, granulocytes, monocytes and macrophages in B-Rag2 KO mice were significantly increased. The results demonstrate that knockout of Rag2 gene has blocked B and T cell development and differentiation in spleen. Values are expressed as mean ± SEM.
Analysis of T cell subpopulations in spleen
Analysis of splenic T cell subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-Rag2 KO mice (n=3, 6-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 TCRβ+ T cell population and used for further analysis as indicated here. (B) Results of FACS analysis. The percent of CD8+ T cells, CD4+ T cells and Tregs in B-Rag2 KO mice were significantly decreased compared to those in wild-type C57BL/6 mice, demonstrating that knockout of Rag2 gene has blocked T cell development and differentiation in spleen. Values are expressed as mean ± SEM.
Analysis of immune cell subpopulations in thymus
Analysis of thymic immune cell subpopulations by FACS. Thymuses were isolated from female C57BL/6 and B-Rag2 KO mice (n=3, 6-week-old). Flow cytometry analysis of the thymocytes was performed to assess immune cell subpopulations. (A) Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. (B) Results of FACS analysis. Compared to the wild-type C57BL/6 mice, percent of total T cells, CD4+ T cells and CD8+ T cells were significantly decreased in B-Rag2 KO mice, while the percent of other cells including B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in B-Rag2 KO mice were similar to those in the C57BL/6 mice. Results demonstrate that knockout of Rag2 gene has blocked T cell development and differentiation in thymus. Values are expressed as mean ± SEM.
Analysis of T cell subpopulations in thymus
Analysis of thymic T cell subpopulations by FACS. Thymuses were isolated from female C57BL/6 and B-Rag2 KO mice (n=3, 6-week-old). Flow cytometry analysis of the thymocytes was performed to assess T cell subpopulations. (A) Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ 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 not detectable in homozygous B-Rag2 KO mice. Results demonstrate that knockout of Rag2 gene has completely blocked T cell development and differentiation in thymus. Values are expressed as mean ± SEM.
Analysis of immune cell subpopulations in blood
Analysis of blood immune cell subpopulations by FACS. Blood were isolated from female C57BL/6 and B-Rag2 KO mice (n=3, 6-week-old). Flow cytometry analysis of the blood cells was performed to assess immune cell subpopulations. (A) Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. (B) Results of FACS analysis. Compared to cells in wild-type C57BL/6 mice, percent of total T cells, CD4+ T cells, CD8+ T cells and B cells were significantly decreased, while percent of NK cells, monocytes and macrophages in B-Rag2 KO mice were significantly increased. Percent of dendritic cells and granulocytes in B-Rag2 KO mice were similar to those in wild-type C57BL/6 mice. The results demonstrate that knockout of Rag2 gene has blocked B and T cell development and differentiation in blood. Values are expressed as mean ± SEM.
Analysis of T cell subpopulations in blood
Analysis of blood T cell subpopulations by FACS. Blood were isolated from female C57BL/6 and B-Rag2 KO mice (n=3, 6-week-old). Flow cytometry analysis of the blood cells was performed to assess T cell subpopulations. (A) Representative FACS plots. Single live CD45+ cells were gated for TCRβ+ T cell population and used for further analysis as indicated here. (B) Results of FACS analysis. Percent of CD4+ T cells, CD8+ T cells and Tregs in B-Rag2 KO mice were significantly decreased compared to those in the C57BL/6 mice, demonstrating that knockout of Rag2 gene has blocked T cell development and differentiation in blood. Values are expressed as mean ± SEM.
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B-Rag2 KO mice exhibit the significantly reduced lymph node size and weight
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The size and weight of lymph nodes of B-Rag2 mice were significantly reduced compared to that of wild-type C57BL/6 mice. Lymph nodes of inguen and axilla were isolated from female wild-type C57BL/6 mice and B-Rag2 KO mice and weighed (n=3, 8-week-old). The size and weight of lymph nodes isolated from B-Rag2 KO mice were significantly reduced compared to that of wild-type C57BL/6 mice.
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Summary
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Leukocyte subpopulation analysis：
- Thymus: Knockout of Rag2 gene has blocked T cell development and differentiation in thymus.
- Spleen: Knockout of Rag2 gene has blocked B and T cell development and differentiation in spleen.
- Blood: Knockout of Rag2 gene has blocked B and T cell development and differentiation in blood.
The size and weight of lymph nodes:
- The size and weight of lymph nodes isolated from B-Rag2 KO mice were significantly reduced compared to that of wild-type C57BL/6 mice.
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