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Production of rBVRA and hBVRA containing cell lysates

RD Remco van Dijk
SA Sem J. Aronson
DW Dirk R. de Waart
SG Stan F. van de Graaf
SD Suzanne Duijst
JS Jurgen Seppen
RE Ronald Oude Elferink
UB Ulrich Beuers
PB Piter J. Bosma
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HEK293T cells were seeded in a T162 cm² flask and upon reaching 30–40% confluence lentivirus containing the hBVRA or rBVRA transgene was added in the presence of 10 μg/ml DEAE Dextran. Four hours thereafter, the medium was refreshed with complete DMEM. 48 hours after transduction the cells were washed and harvested in ice-cold PBS. Cells were then pelleted at 1500 rpm at 4 °C for 10 minutes and taken up in a hypertonic buffer (10 mM HEPES, 40 mM KCl, 2 mM MgCl2, 10% gycerol). After 15 minutes on ice, 25 stokes with a dounce homogenizer were used to break the cells. To remove cell membranes and debris, the homogenate was centrifuged at 80.000 RCF (xg) for 1 hour at 4 °C and the supernatant was harvested, aliquoted and stored at −80 °C.

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