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Making a recombinant baculovirus using the flashBAC system

OET will prepare recombinant baculoviruses using the flashBAC system.  We need to be supplied with purified transfer plasmid containing the gene of choice (either from yourselves or via our cloning service).  We also need information to prepare an ACGM risk assessment at Level 1, so we need details of the name and nature of the protein to be expressed and an assurance that the protein is not toxic or harmful to humans as we are only able to carry out ACGM Class 1 projects.  In order to help you provide the information we need, please complete the Fact Sheet and return it to us.

We offer a choice of three flashBAC vectors:

  1. The original flashBAC vector suitable for most projects where the gene to be expressed is likely to be simple and expressed in the cytoplasm or nucleus of infected cells.
  2. flashBACGOLD that provides superior levels of expression for any protein that is destined for secretion or to be inserted in the membrane, or for any protein that might be particularly liable to degradation.  This expression vector has been modified by deletion of the chitinase gene, that impairs the function of the secretory pathways in virus-infected cells, and the cathepsin gene which is a viral protease that can cause degradation of recombinant proteins that happen to contain the target sites for proteolysis.
  3. flashBACULTRA contains deletions of the chitinase and cathepsin genes, as above, but also deletion of the p10 gene, that provides the best choice for the most difficult to express proteins.

OET will prepare a recombinant virus by co-transfecting insect cells (Spodoptera frugiperda Sf9) with the transfer plasmid andflashBAC DNA.  Use of the flashBAC systems means that we can easily prepare multiple recombinant viruses and we offer a service to make up to 24 at a time using a semi-robotic platform.

Five days after transfection, the culture medium will contain recombinant virus that will be harvested and used to amplify a 50 ml seed stock of virus in Sf9 cells in serum-free medium.  The seed stock of virus will be titrated using either plaque-assay or baculoQUANT to determine the virus titre.  The virus stock will be stored in the dark at 4 C.

OET will carry out an initial, quick test for expression by infecting dishes of Sf9 cells with seed stock virus (200 ul); a dish of control cells will also be mock-infected with culture medium.  At 48 hours post-infection, the cells and culture medium will be harvested and analysed separately for expression of protein by Western Blot using either anti-tag anti-serum (usually anti-His-tag) or using primary antisera supplied by the client or purchased commercially by OET (on the instructions of, and at cost to, the client).

The results will be discussed with the client prior to any further service work being undertaken, or if the project ends at this stage, the virus will be shipped to the client and a full report of the work carried out is sent via email.

Please contact us for further details