American Society of Hematology

Fig. 3.

$Fig. 3. Reconstitution of rag1+/gfp+ spleen cells into rag1 knockout mice. / (A) Site of RAG1 expression in the reconstituted mice. Inducibility of RAG1/GFP signal was examined by confocal laser microscopy and flow cytometry with heterozygous rag1+/gfp+spleen cells (9-week-old mice) reconstituted into rag1knockout mice. Ten million splenocytes from heterozygousrag1/gfp knockin mice were injected into rag1knockout mice intravenously. These mice were identified as GFP-SPL. After 14 days, mature lymphocytes resided in the peripheral lymphoid organs of GFP-SPL reconstituted rag1 knockout mice. These reconstituted mice were then used for intraperitoneal Ag immunization with DNP-KLH (100 μg) in CFA. The fluorescence signal was compared with PNA staining on an adjacent serial section for WT and GFP-SPL mice. Original magnification, 100 ×. (B) Flow cytometric analysis of GFP signal. On days 8 and 16, spleen cells from WT, GFP-SPL, and RAG1/GFP mice were characterized by flow cytometry using PE–anti-B220 mAb and biotinylated–-IgM mAb plus streptavidin-R670. Representative results on day 16 after Ag immunization are shown. GFP fluorescence in B220+ sIgM+ fractions of WT (G1), GFP-SPL (G2), and RAG1/GFP nonimmunized control (G3) are compared in the histogram. (C) Immune response to TD-Ag. Serum samples were also kept and examined using an ELISA method that detected Ag-specific Ab of IgM and IgG1 classes. GFP-SPL mice with or without Ag immunization showed the Ag-specific response.$

Reconstitution of rag1⁺/gfp⁺ spleen cells into rag1 knockout mice.

(A) Site of RAG1 expression in the reconstituted mice. Inducibility of RAG1/GFP signal was examined by confocal laser microscopy and flow cytometry with heterozygous rag1⁺/gfp⁺spleen cells (9-week-old mice) reconstituted into rag1knockout mice. Ten million splenocytes from heterozygousrag1/gfp knockin mice were injected into rag1knockout mice intravenously. These mice were identified as GFP-SPL. After 14 days, mature lymphocytes resided in the peripheral lymphoid organs of GFP-SPL reconstituted rag1 knockout mice. These reconstituted mice were then used for intraperitoneal Ag immunization with DNP-KLH (100 μg) in CFA. The fluorescence signal was compared with PNA staining on an adjacent serial section for WT and GFP-SPL mice. Original magnification, 100 ×. (B) Flow cytometric analysis of GFP signal. On days 8 and 16, spleen cells from WT, GFP-SPL, and RAG1/GFP mice were characterized by flow cytometry using PE–anti-B220 mAb and biotinylated–-IgM mAb plus streptavidin-R670. Representative results on day 16 after Ag immunization are shown. GFP fluorescence in B220⁺ sIgM⁺ fractions of WT (G1), GFP-SPL (G2), and RAG1/GFP nonimmunized control (G3) are compared in the histogram. (C) Immune response to TD-Ag. Serum samples were also kept and examined using an ELISA method that detected Ag-specific Ab of IgM and IgG1 classes. GFP-SPL mice with or without Ag immunization showed the Ag-specific response.

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