Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Engineering a BCR-ABL-activated caspase for the selective elimination of leukemic cells

doi: 10.1073/pnas.1206551110

Figure Lengend Snippet: iCaspase-8 selectively kills BCR-ABL–positive mouse HSCs. (A) Mouse KLS cells were cotransduced with BCR-ABL (MSCV-IRES-GFP) and iCapsase-8 (MSCV-IRES-YFP-WRE) or empty vector. BCR-ABL was either wild type (WT) or carried an imatinib-resistant mutation (E255K, T315I, M351T). GFP-YFP double-positive cells were further selected by FACS and plated in methylcellulose medium with or without 2 µM imatinib. The colony numbers were counted 7 d after plating (*P < 0.05 YFP vs. iC8 by Student t test). (B) Mouse KLS cells were transduced with iCapsase-8 (MSCV-IRES-GFP-WRE) or empty vector. GFP positive cells were selected by FACS and plated in methylcellulose medium. The colony numbers were counted 7 d after plating. Averages with SEM are shown.

Article Snippet: iCaspase-8, iCaspase-9, iApoptosome, and iDISC were cloned into MSCV vectors that contain the WRE element and a bicistronic IRES-GFP or -YFP (MSCV-IRES-GFP-WRE or MSCV-IRES-YFP-WRE) (Addgene plasmid 12282; ref. 24 ).

Techniques: Plasmid Preparation, Mutagenesis, Transduction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Engineering a BCR-ABL-activated caspase for the selective elimination of leukemic cells

doi: 10.1073/pnas.1206551110

Figure Lengend Snippet: iCaspase-8 selectively kills blast crisis CML cells in mouse. (A) Lin− BCR-ABL+ NUP98-HOXA9+ cells isolated from mice with blast crisis CML were infected with control vector or iCaspase-8 (MSCV-IRES-GFP-WRE). Forty-eight hours after infection, GFP and YFP (BCR-ABL) coexpressing cells were selected and plated in methylcellulose medium for colony forming assays. The colony numbers were counted 7 d after plating. Average colony numbers with SEM are shown (*P < 0.05 by Student’s t test). (B) Cells infected with BCR-ABL, NUP98-HOXA9, and either control vector or iCaspase-8 were transplanted and survival was monitored (control, n = 23; iCaspase-8, n = 20; *P < 0.05 by Kaplan–Meier analysis with log-rank test). (C and D) Spleens were collected from each mouse and weighed (C). Average weights and SEM are shown (*P < 0.05 by Student’s t test). Representative spleens are shown in D: spleens from control (Left) and iCaspase-8 mice [surviving mice (Center) and leukemic mice (Right)].

Article Snippet: iCaspase-8, iCaspase-9, iApoptosome, and iDISC were cloned into MSCV vectors that contain the WRE element and a bicistronic IRES-GFP or -YFP (MSCV-IRES-GFP-WRE or MSCV-IRES-YFP-WRE) (Addgene plasmid 12282; ref. 24 ).

Techniques: Isolation, Infection, Control, Plasmid Preparation