in vitro

Sunday, 20 March, 2022

Lentivirus packaging

 

Lentiviruses are classified under BSL2, because it can infact human cells

 

BSL2 requirements:

-biosafety cabinet (operator safety and sterility)

-PPE (mask, gloves, labcoat, shoes)

 

before packaging need to be determined:

1.      packaging cell line ( industrial standard: 293T cells)

2.      titer (supernatant collection produce 106-7  IU/ml, higher titer can be achieved with transfecting cells at a larger scale with extra concentration steps)

 

-subculture cells (70-75% confluence at the time of transfection) must be healthy, no clamps

-lentivirus (gene of interest) and packaging plasmid (viral packaging components) need to be cotransfected

-quantifiable transfection efficiency (for example checking fluorescence if reporter was inserted)

-supernatant (lentivirus particles) need to be collected 72 h after transfection in a sterile bottle and store at 4°C (bottles should be made from polypropylene or silicone and not from hydrophobic plastic like polysytrene)

-if more viral particals is needed, perform second collection and combine the second with the first harvest 

-0,45um filter is recommended to remove all cell debris from the viral particles

dispose the cells according the biosafety guideline (bleach and/or autoclaving)

-collected virus can be stored at 4°C for a week. For long-term storing it should be frozen at -80°C, but should be avoided freeze/thaw cycles (aliquotes)

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viral constructs