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2.3.11.1 Transduction setup and spinfection

Aliquots of patient-derived peripheral blood or bone marrow blasts were

retrieved from liquid nitrogen either on the day of, or the day prior to spinfection, thawed and counted with Trypan Blue to assess viability as described in 2.3.1. Cells were centrifuged at 300 x g, 5 mins and resuspended at 1 x 106 cells/ml in either αMEM supplemented with 10% v/v FCS, 10% v/v horse serum, 10% v/v HS-5 conditioned medium, see 2.3.1.5, or in serum free expansion medium (SFEM) supplemented with 10% v/v FCS. Five hundred microliters (5 x 105 cells) were pipetted into wells of a 48 well culture plate and supplemented with the appropriate polybrene or protamine. For some experiments additional cytokines were added to specific wells to assess their effect on transduction and cell survival. These included stem cell factor, 50 ng/ml, interleukin 7 (IL-7), 10 ng/ml and IL-3, 20 ng/ml. SLIEW/SLMIEW lentivirus was added to the wells, with one well left without virus to provide an untransduced control. The plate was balanced to within 0.2 g and sealed with Parafilm.

Spinfection was performed as detailed in section 2.3.9.5.

Following overnight culture, 350 µl transduction medium was aspirated from the cells and replaced with 850 µl fresh medium. For those specimens transduced in the presence of cytokines, the fresh medium was supplemented with the same cytokine concentrations. Cells were either transplanted into NSG mice, see 2.3.11.4, or analysed my five-colour flow cytometry on day 3 or 4 following transduction, see 2.3.11.2.

2.3.11.2 Assessment of transduction by multicolour flow cytometry Analysis of transduction was performed by flow cytometry using either single colour assessment of EGFP (FACS Calibur, 2 samples only) or four-colour assessment of EGFP, anti-CD19 PE, anti-CD20 PerCP Cy5.5, anti-CD34 APC. For samples which had been harvested following xeno-transplantation, murine cells were excluded by staining with anti-murine CD45/Ter119 PE Cy7.

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Cells were resuspended and washed once in 3 ml PBS in a flow cytometry tube, 450 x g, 4 minutes. Cells were resuspended in 100 μl PBS and stained with anti-CD19, 10 μl, anti-CD20, 10 μl, anti-CD34, 2.5 μl with or without anti-murine CD45, 2.5 μl, anti-Ter119, 2.5 μl, at room temperature, protected from light for 20 minutes. Cells were then washed twice in 2.5 ml PBS, 450 x g, 4 minutes and resuspended in 300 μl PBS ahead of analysis using a FACS Canto II flow cytometer.

2.3.11.3 Washing procedure for removal of residual lentivirus In order to remove residual, infectious lentivirus, transduced cells were stringently washed each day from day 1 post-transduction onwards. This protocol was developed during the course of this project.

On Day 1 following transduction, 350 μl supernatant was removed from the transduced cells. Cells were resuspended in 850 μl medium followed by the addition of a further 20 ml RPMI 1640, 10% FCS. Cells were centrifuged 300 x g for 10 minutes. The supernatant was aspirated using a plastic pipette (to avoid the risk of sharps related injuries) and the cells resuspended to repeat the above wash procedure. Following the second wash, cells were resuspended in 1 ml SFEM supplemented as for the transduction experiment. The wash

procedure was repeated on subsequent days.

2.3.11.4 Xenotransplantation studies using NSG mice

Xenotransplantation studies were conducted using intrafemoral injection into NOD/scid IL-2 receptor gamma common chain knockout (NSG) mice (NOD.Cg- Prkdcscid Il2rgtm1Wjl/SzJ) mice (Shultz, Lyons et al. 2005) (Jackson Laboratories, supplied by Charles River UK Ltd). These mice, in addition to the severe

combined immune deficiency (scid) background, have a further deletion of the IL-2 receptor gamma common chain. The product of this gene is an intrinsic component of a number of cell surface receptors within the adaptive immune system and these mice therefore have profound immune deficiency.

Functionally they lack B- and T-cells, NK cells and have disorders of

macrophage and complement function. In addition to currently providing the most sensitive model for leukaemic engraftment (McDermott, Eppert et al.

2010), NSG mice also have a longer lifespan than NOD/scid mice and are therefore well suited to transplantation studies which can take many months. Intrafemoral transplantation was performed by one of three trained members of the laboratory. Transplants were performed in a laminar flow hood with aseptic technique, under general anaesthesia. Mice received subcutaneous Carprofen analgesia, 5 mg/kg, whilst anaesthetised. A suspension of patient derived leukaemic cells or derived cell line cells of between 20 – 30 μl was injected into the femur through a hole made in the distal femoral joint, as described in (le Viseur, Hotfilder et al. 2008).

2.3.11.5 In vivo imaging of transduced, fLuc+ leukaemia cells

In vivo imaging of mice transplanted with leukaemic blasts (patient-derived or cell line) transduced with SLIEW or SLMIEW viruses was performed using an IVIS Spectrum bioluminescent camera. For standard planar imaging, mice received an intraperitoneal injection of 3 µg D-Luciferin. For 3 dimensional imaging, mice received 4.5 µg D-Luciferin. Mice were anaesthetised using 3% v/v isofluorane in an induction chamber so that imaging could begin, with on- going 2% v/v isofluorane, 10 minutes after the D-Luciferin injection.

2.3.11.6 Harvesting mice for engraftment leukaemic material

At the end of an experiment, mice were killed by cervical dislocation. Material was harvested from the spleen and bone marrow, and for one experiment also from the liver and kidney (collected as for the spleen). The spleen was

dissected out of the abdomen, weighed and macerated through a 40 μm cell strainer into PBS. Femurs and tibias were dissected out of both legs, the end cut off and the marrow flushed out with PBS using a 25G needle. Bone marrow was also then passed through a 40 μm cell strainer to provide a single cell suspension and remove any fragments of bone.

2.3.11.7 Harvesting organs for immunohistochemistry

For one mouse transplanted with L578-SLIEW, the brain, kidneys, liver and spleen were dissected out and placed into formalin for fixation. Organs were then embedded in paraffin and cut into 4 μm sections and mounted on glass

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slides. Haematoxylin and eosin staining was performed by Dr Jennifer Jackson, Northern Institute for Cancer Research. Immunohistochemical staining was performed commercially by the Department of Pathology, Royal Victoria Infirmary using a Ventana Benchmark XT or Ventana Benchmark Ultra and Ventana Ultraview detection system (CD10, CD79a, TdT) and University College London (CD19).

Slides for fluorescent microscopy were mounted using DAPI mount by Dr J Jackson and viewed using a Leica DSM fluorescence microscope and captured with SPOT RT digital CCD camera.

2.3.12 Cloning of a single vector combining in vitro analysis, in vivo