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Lentiviruses are a part of the retrovirus family, and in this project pSIEW vector backbone (Empty, RUNX1-ETO, RUNX1-EVI-1 and DNCEBP) produces lentiviral particles. Packaging and envelope genes are on a separate plasmid to prevent further virus particle generation once transduced into the target cell. In

this project we also used CEBPA-ER plasmid to produce γ-retrovirus particles, and these require different viral packaging plasmids and transduction method as will be described below.

2.14.1 Transfection of HEK293T cells for lentiviral production

HEK293T cells were re-plated 24 hour prior to transfection, so that at time of transfection they were 80-90% confluent. On the day of transfection TransIT- 293T (Mirius, USA) was brought to room temperature. Trans-IT-293T forms a complex with DNA plasmids to enable transfection into cells. A DNA mix was made from plasmids (Backbone vector containing transgene 30µg, and the packaging vectors: Tat 1.2µg, Rev 1.2µg, Gag/Pol 1.2µg, VSV-G 2.4µg (gift from George Murphy, Boston, USA) (Sommer et al., 2009). For each 15cm2

2ml of Optimem serum free media was mixed with 90µl of TransIT-293 (Mirius, USA). This was allowed to rest at room temperature for 15 minutes. DNA mix was added to the TransIT-293 mixture and was left at room temperature for a further 15 minutes. Fresh DMEM with 10% FCS supplemented with glutamine and penicillin/streptomycin was exchanged with previous media on the HEK293T plates. The TransIT-293 – DNA mixture was then added dropwise to the HEK293T plate. Viral supernatant was collected after 48 hours and subsequently every 12 hours for 36 hours.

2.14.2 Transfection of HEK293T cells for CEBPA-ER virus

production

HEK293T cells and TransIT-293T (Mirius, USA) was prepared as above. DNA mix was made from plasmids (Backbone vector containing transgene 36µg, Tat 1.2µg, Gag/Pol 30µg, Env 9µg (gift from James Mulloy, Cincinnati, USA) and was sufficient for 3x 10cm2 dish. For each 10cm2 dish 1.5ml of Optimem serum free media was mixed with 75µl of TransIT-293. This was allowed to rest at room temperature for 15 minutes. DNA mix was added to the TransIT-293 mixture and was left at room temperature for a further 15 minutes. Fresh DMEM with 10% FCS supplemented with glutamine and penicillin/streptomycin was exchanged with previous media on the HEK293T plates. The TransIT-293

– DNA mixture was then added dropwise to the HEK293T plate. Viral supernatant was collected after 48 hours and subsequently every 12 hours for 36 hours.

2.14.3 Virus concentration

The virus concentration technique was the same for all viral particles. Viral supernatant was centrifuged at 1660xg 4°C 15 minutes to pellet cell debris. The supernatant was then filtered through a 0.45m disc filter.

The viral supernatant was concentrated using ultracentrifugation 120000xg for 2 hours 4°C in a SW28 rotor in an OptimaTM _{100k-XL centrifuge (Beckman}

Coulter, USA). The supernatant was poured off and the viral pellet was re- suspended with the residual fluid. Alternatively the viral supernatant was concentrated using a Centricon Plus-70 100kDa filter (Millipore, USA), using the manufacturer’s instruction. The column was pre-rinsed with sterile water and the column centrifuged at 2000xg, 25 minutes at 4 degrees. The column was then inverted and the concentrate recovered by centrifugation at 1000xg for 2 minutes.

2.14.4 Titration of viruses on HEK293T cells

Viruses were titrated before transduction of CD34+ PBSCs. 1x105 _HEK293T

cells were plated in each of 24 well plates 24 hours prior to transduction. On day of transduction viral concentrate was serially diluted 1:10 with media and media in the wells replaced with 450l of fresh media. 50l of the concentrate and subsequent 1:10 serial dilutions was added to the HEK293T cells. Polybrene at a final concentration of 8g/ml was also added prior to spinoculation at 1500xg for 2 hours at 32°C. The principle of spinoculation is that through centrifugal force, viral particles and the cells due for transduction co-localise, thereby increasing the odds of viral entry into the cells. The plate was subsequently returned to the incubator overnight and at the next morning the viral media was removed and exchanged with fresh media. Viral transduction was estimated by eGFP percentage by flow cytometry 5 days after

viral transduction. Cells were washed with PBS and fixed with 1% formaldehyde for 10 minutes at room temperature. After washing again, the cells were detached by washing with PBS and taken to the flow cytometer. Dilution points with 1-20% eGFP positive cells were used to estimate the viral titre. At least an average of two dilutions is required for an accurate estimate. eGFP-titre formula: Transducing Units/ml = (F × N)/ (100 × V × Df),

F=percentage of eGFP positive cells (in %)

N=number of cells spinoculated (1x105 in this example)

V= Volume of diluted viruses added to each well (50l in this example)

Df=dilution factor of the viral concentrate at to each wells (x1 for the first well in this example)

2.14.5 Lentiviral transduction of CD34+ PBSCs and SKH-1

Cryopreserved CD34+ PBSCs were thawed with warmed IMDM with 20% BIT9500 serum substitute. The CD34+ PBSCs were incubated overnight in IMDM with serum substitute with cytokines and SR1 mix as described above in section 2.4.

CD34+ were transduced with viral concentrates (at a MOI of up to 36) with polybrene at 4g/ml by spinoculation at 1500xg for 2 hours at 32°C in non- tissue culture treated plates. The plate was subsequently returned to the incubator overnight and at the next morning the viral media was removed and exchanged with fresh media. Viral transduction was estimated by eGFP percentage by flow cytometry 5 days after viral transduction. Cell sorting by FACS was undertaken by staining with either CD34-PE or CD34-APC (both Miltenyl Biotech, UK) and corresponding isotype control.

SKH-1 cells were transduced with lenitviruses (pSIEW empty vector or pSIEW DNCEBP) in the same manner, except polybrene was used at 8µg/ml. Cell sorting for GFP positive cells was undertaken by FACS.

2.14.6 Retroviral transduction with Retronectin

CEBPA-ER vector required RectroNectin (recombinant human fibronectin

fragment, Takara, Japan) assisted transduction. RectroNectin has two domains that allow binding to a target cell expressing the integrin VLA-4 or VLA-5, but also has a third domain that enables binding to viral particles

((http://www.clontech.com/GB/Products/Viral_Transduction/Hematopoietic_Cell _Transduction/ibcGetAttachment.jsp?cItemId=61561&fileId=6521143&sitex=10 030:22372:US), accessed 3rd September, 2016). Non-tissue culture treated 6 well plate was incubated with 2ml RetroNectin solution in PBS at 24µg/ml for 2 hours at room temperature. The RectroNectin solution was subsequently removed and the plate blocked with 2ml PBS with 2% BSA for 20minutes at room temperature. Finally, the PBS/BSA solution was removed and the wells washed with 2ml Hank Balanced Salt solution containing 2.5% HEPES.

Concentrated virus was coated onto the wells by centrifugation for 45 minutes at 2000xg at room temperature. Virus loading was repeated once more before 1x106 _{SKH-1 cells, in media mixed with concentrated virus (50:50 ratio), at a}

final concentration of 0.5x106 _{/ml was added to the wells with polybrene at}

8µg/ml. The plate was left overnight at 37ºC, 5% CO2 in a humidified incubator.

On the next day, the cells were centrifuged at 300xg at room temperature for 5 minutes and the cells were re-suspended in standard RPMI 1640 with 10% FCS, supplemented with penicillin, streptomycin and glutamine. CEBPA-ER transduced SKH-1 cells were isolated by FACS using GFP labelling.