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Figure 2 | Retrovirology

Figure 2

From: Mouse mammary tumor virus-based vector transduces non-dividing cells, enters the nucleus via a TNPO3-independent pathway and integrates in a less biased fashion than other retroviruses

Figure 2

Transduction of non-dividing cells. (A) 293 cells were exposed to ionizing radiation (50 Gy) and 24 h later (at the time of transduction) stained with propidium iodide and analysed by flow cytometry. Non-irradiated cells used as a control showed a well defined G1 peak. Exposure to radiation resulted in an accumulation of virtually all cells in the G2/M stage of the cell cycle. (B) The irradiated and non-irradiated cells were transduced with MMTV-based vector. As positive and negative controls, for transduction of non-dividing cells, vectors derived from HIV-1 and MLV were used. The transduced cells were analysed by UV microscopy and FACS analysis 3 days post transduction. (C) Viral titers expressed as percentages of titers obtained on dividing cells (black columns). The cells of one set of transduced irradiated cells were exposed to AZT (10 μM, white columns). Another control is represented by a heat-treated vector (65°C, 10 min, grey columns). For the controls, the titer is given as a percentage of titers observed on untreated cells. Error bars indicate standard deviation from the mean of three independent transduction experiments. (D) The irradiated cells were infected either with MMTV(GR) or the same virus treated with heat (65°C, 10 min). The infected cells were cultured in the absence or presence of AZT (GR + AZT) for 7 days. MMTV-specific PCR was used to detect the presence of MMTV provirus in the infected cells. GAPDH-specific PCR with equivalent DNA amounts was used as control. (E) The purity of bone marrow-derived CD34+ preparations was verified by flow cytometry using an anti-CD34-PE antibody. (F)+ (G) The hematopoietic progenitor cells (1 × 106 cell/ml) transduced with the MMTV-or HIV-based vector (1 × 107 TU/ml) were cultured in the presence or absence of growth factors for 16 days and analysed by UV microscopy and FACS. Representative figures of microscopic (G) and flow cytometric (F) analysis of transduced cells are shown.

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