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Blood, Vol. 112, Issue 12, 4466-4474, December 1, 2008
Interfering RNA-mediated purine analog resistance for in vitro and in vivo cell selection
Blood Porter and DeGregori
112: 4466
Supplemental materials for Porter and DeGregori
Files in this Data Supplement:
- Figure S1. Knockdown of HPRT provides hematopoietic cells with resistance to 6MP (JPG, 50.5 KB)
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Transduced and puromycin selected FL5.12 cells were cultured at an initial concentration of 5 × 104 cells/ml in the presence of 6MP at 10, 20, 40, 60, and 100nM or no drug (UT). The concentration of live cells after 96 hours is depicted as a percentage of Ctrl UT live cells (p=0.0004; ANOVA).
- Figure S2. Knockdown of HPRT protects cells from G2/M checkpoint arrest and apoptosis induced by 6TG but not cisplatinum or IL3 deprivation (JPG, 174 KB)
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FL5.12 cells transduced with Ctrl or 491 (or mock transduced) were cultured in the presence of 6TG 10nM, cisplatinum 1 µM, no drug (UT), or in the absence of IL3. (A) At 48 hours, an aliquot of the culture was fixed, stained with PI, and analyzed by flow cytometry. Representative histograms of flow cytometry for cell cycle analysis that correspond with data in Figure 4A are shown. (B) The percentage of cells in the G2/M phase of the cell cycle (as defined by 4N DNA content) is graphed. (C) In a separate experiment, after 48 hours, an aliquot of the culture was stained with FITC linked Annexin V and PI and analyzed by flow cytometry. Representative histograms corresponding to data in Figure 4C are shown. (D) The percentages of apoptotic (Annexin V+-PINeg) cells are graphed.
- Figure S3. HPRT knockdown and 6TG treatment potently select for gene modified hematopoietic progenitor cells in mice (JPG, 85 KB)
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Primary BM cells were harvested from donor mice, transduced with CtrlG and 491G vectors, and transplanted into recipient mice. Peripheral blood was periodically analyzed by flow cytometry. The percentage of GFP+ Gr1+ peripheral blood cells from individual recipients of 491G transduced BM is plotted vs. time. Note log scale. Light gray shading indicates background level of GFP detection from negative controls and dark gray shading indicates the lower limit of detection. (A) Percentages of GFP+Gr1+ cells from high/moderate dose experiment. The number of 6TG treated mice at weeks 14 and 16 in the high/moderate dosing experiment is 4 and 3 respectively, due to toxicity related deaths. (B) Percentages of GFP+Gr1+ cells from low dose experiment. Primary recipients numbered 991 and 997 demonstrated most substantial contributions to multi-lineage hematopoiesis in secondary recipients (Table 1).
- Figure S4. Control transduced cells are not selected in vivo after transplantation and 6TG treatment (JPG, 60.5 KB)
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Primary BM cells were harvested from donor mice, transduced with CtrlG vector, and transplanted into recipient mice. Peripheral blood was periodically analyzed by flow cytometry. The percentage of GFP+ cells in Gr1+ (top) and B220+ (bottom) peripheral blood cell populations from recipients of CtrlG transduced BM is plotted vs. time. Note log scale. Five day courses of treatment are indicated by open boxes (0.25mg/kg/d), black boxes (2mg/kg/d), or gray boxes (0.5mg/kg/d). CtrlG transduced mice did not tolerate the moderate dosing strategy due to impaired hematopoiesis and were euthanized without further flow cytometry analysis.
- Figure S5. Gating schemes for identification of HSC and MPP (JPG, 256 KB)
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BM from a subset of PBS and 6TG treated recipients of 491G transduced BM was analyzed by flow cytometry. (A) Lineage/Flk-2 negative cells were analyzed for c-Kit and Sca-1 expression. c-Kit+Sca-1+Lin/Flk-2Neg cells are highly enriched for HSC activity.37 Examples of negative control, 491G PBS, and 491G 6TG treated recipients are depicted. (B) Lineage/CD48 negative cells were analyzed for CD150 and CD244 expression. The CD150+CD244NegLin/CD48Neg population is highly enriched with long-term, multipotent repopulating (HSC) activity, while the CD150NegCD244+Lin/CD48Neg population is enriched for shorter-term multipotent progenitors (MPP).36 Examples of negative control, 491G PBS, and 491G 6TG treated recipients are depicted.
- Figure S6. 6TG affects the frequency and number of phenotypic HSC (JPG, 49.9 KB)
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Wild-type mice were treated with PBS or 6TG using the high and moderate dosing strategies described in the text. At the indicated time-points, the bone marrow was harvested from the tibiae and femurs, counted using propidium iodide exclusion, and analyzed by flow cytometry for HSC (CD150+CD244NegLin/CD48Neg). The number of HSC harvested per mouse is depicted graphically.
- Figure S7. HPRT knockdown and 6TG treatment in primary recipients enhances engraftment of transduced cells in secondary recipients (JPG, 139 KB)
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BM from primary recipient mice treated with PBS or low dose 6TG was harvested and transplanted into 3 sub-lethally irradiated secondary recipients per donor. (A) Peripheral blood was analyzed by flow cytometry. The p-values reflect analyses comparing individual recipients of BM from PBS or 6TG treated donors at 4 (n=12 per group) and 8 (n=9 per group) weeks after transplantation (closed diamonds). The percentages of GFP+ cells from donor mice at week 16 after primary transplantation are also depicted (open boxes). Light gray shading indicates background level of GFP detection from negative controls and dark gray shading indicates the lower limit of detection. (B) Representative histograms of flow cytometry for GFP expression in secondary recipients (Table 1 and Figure S7B).
- Figure S8. Sequences coding for shRNA against human Chk2 and murine HPRT (JPG, 72.7 KB)
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