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Lentiviral RNAi Protocols: Biosafety Issues


The biosafety office at your institution must be notified prior to use of this system for permission and for further institution-specific instructions. BL2 conditions should be used at all times when handling the virus. All decontamination steps should be performed using 70% ethanol/1% SDS.Gloves should be worn at all times when handling lentiviral preparations, transfected cells or the combined transfection reagent. Just remember that although this virus has been significantly modified for biosafety, it derived from HIV and with a VSV pseudotype human cells can be infected even if they are not dividing. That said the following modifications have been made to prevent viral replication.

  1. Packaging vector lacks both LTRs and has no viral packaging signal (y)

  2. The following viral genes have been deleted from the packaging vector: env, tat, rev, vpr, vpu, vif and nef.

  3. Rev is supplied in trans on a different vector (RSV-Rev).

  4. The vector expressing the packaged viral genome has a self-inactivating LTR (TATA box deletion) and expresses no viral gene products.

  5. Envelope, in this case VSVG, is expressed on a separate vector.

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