Expression of RAG1/GFP signal in the peripheral lymphoid organs.

(A) Flow cytometric analysis of the GFP expression in B220⁺cells of the spleen, Peyer patch, axillary LN, and mesenteric LN of heterozygous rag1/gfp knockin mouse (left). Five percent of splenic B cells expressing the highest level of GFP are shown in the gated area on the left of panel A. Next, splenic B220⁺ B cells were separated into GFP⁻, GFP^low, and GFP^high populations by cell sorter using gates shown in the left panel. The RNA isolated from each cell population was used for reverse transcriptase–PCR (RT-PCR) with gene specific primers for RAG1, GFP, and HPRT, respectively (right). Only the GFP^highB cells showed rag1 mRNA. (B) Flow cytometric analysis of the splenic B cells. GFP signal was compared in B220⁺ cell fractions further separated by staining with sIgM (G1, G2, G3, and G4), CD43 (G5 and G6), and HSA⁺ (G7 and G8). GFP signals in splenic B cells were also analyzed in different fractions stained with sIgM⁺ and sIgD⁺ (T1, T2, and M). For regions G1 to G8, the vertical bars to the right of each histogram show the percentage of B220⁺ cells represented by each gate that were GFP⁺. (C) Fluorescence microscopy analysis of GFP signal. The upper panels show GFP signals of purified splenic cells sorted with B220⁺ from WT or RAG1/GFP knockin mice. The middle panels show tissue sections from thymus and spleen of WT or RAG1/GFP mice analyzed for GFP expression. The bottom panels show the same sections stained with H&E (middle panels). Original magnification, 100 ×.