The deletion of Gfi1b results in increased Rag transcription and gene rearrangement. (A) Quantitative RT-PCR analysis of Rag transcripts in Wt and Gfi1b^−/− AMuLV cell lines. Levels are expressed relative to HPRT transcripts and consist of the mean of triplicate assays. (B) Flow cytometric analysis of Wt and Gfi1b^−/− AMuLV cell lines infected with a rearrangement reporter construct diagrammed above. Upon rearrangement, the IRES-GFP segment is deleted and hCD4 is expressed. Eμ, IgHC enhancer; VHKI, VH gene promoter; triangles, RSSs. Data are representative of two independent experiments. (C) PCR assay for Vκ-Jκ1 coding joints in a series of diluted DNA samples from Wt and Gfi1b^−/− AMuLV cell lines. S, splenic DNA; H2O, water-only control. Data are representative of two independent experiments. (D) LM-PCR analysis of DNA breaks at the Jκ1 RSS in Wt and Gfi1b^−/− AMuLV cells. The inverted image of an agarose gel analysis of PCR products is shown. Data are representative of two independent experiments. (E, left) Quantitative RT-PCR analysis of Rag1 transcript levels (relative to HPRT) in Wt and Gfi1b^−/− AMuLV cell lines treated for the indicated times with STI-571, and, where indicated, washed and cultured for an additional 36h. (E, right) Replot of data shown on the left on a smaller scale to reveal values from the wt cell line. Error bars denote standard deviation of triplicate assays. Data are representative of two independent experiments.