T-ALLs arising from cultured ICN1⁺Arf^+/+ donor cells retain the gene but do not express p19^Arf protein. (A) Leukemic GFP⁺ splenocytes from 8 mice that had received cultured (day-12) thymocyte-derived donor cells of the indicated Arf genotypes (top) were purified by FACS. Genomic DNA prepared from these cells was used as a template for PCR performed with primers specific for exon-1β and exon-2 of the Ink4a-Arf locus. Exon-1β encodes the unique N-terminus of p19^Arf, whereas exon-2 encodes C-terminal portions of p19^Arf and p16^Ink4a from alternative reading frames. DNAs extracted from unmanipulated Arf^+/+ bone marrow cells and from mouse NIH-3T3 cells that had deleted the Ink4a-Arf locus during the process of immortalization were used as the positive and negative controls, respectively. The fact that no exon-1β signal was revealed in samples 7 and 8 generated from Arf^−/− donor cells indicates that the purified leukemic cells were uncontaminated by normal Arf^+/+ cells. (B) Although all genotyped leukemias retained the Arf locus, immunoblotting of electrophoretically separated proteins extracted from robustly GFP-positive lymph nodes taken from the same recipients of Arf^+/+ donor cells failed to reveal p19^Arf expression. Arf expression is also silenced in normal bone marrow (BM) cells. Cells engineered to express Arf conditionally under the control of a metallothionein promoter (MT-Arf cells) were induced with zinc. The immunoblot was developed with a highly sensitive monoclonal antibody generated to mouse p19^Arf (Bertwistle et al³⁴ ), and the film was purposely overexposed in an attempt to reveal p19^Arf expression. Nucleophosmin (NPM), an abundant nucleolar protein, was used as the loading control. (C) PCR analysis as in panel A. Samples 1 to 4 were taken from irradiated recipients that had received a transplant of uncultured ICN1-expressing bone marrow from 5-FU–conditioned donors. Samples 5 to 10 were taken from nonirradiated recipients that had received 5-FU–conditioned bone marrow transduced with ICN1 and cultured on OP9 stroma before their adoptive transfer. Control PCR reactions were performed using DNAs extracted from unmanipulated Arf^+/+ bone marrow cells, NIH-3T3 cells lacking the Ink4a-Arf locus, bone marrow cells from Arf^−/− mice in which exon-1β is disrupted, bone marrow cells from p16^Ink4a-null mice in which the unique Ink4a exon-1α was disrupted, and from mice in which Ink4a-Arf exon-2 was disrupted. (D) Immunoblotting for p19^Arf was performed on bone marrow taken from the mice analyzed in panel C.