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Producing Your Recombinant Baculovirus

Updated: Nov 28, 2023

There are 2 main methods through which you can happily produce recombinant baculoviruses, firstly by using systems based on homologous recombination such as flashBAC™ and BacPAK6. It is also possible to produce your virus using site specific transposition systems such as bac-to-bac.


These methods are covered in more detail below.

Homologous Replication (flashBAC™, BacPAK6):

In this method the gene of interest is first cloned into a transfer vector (Fig. 1) containing sequences that flank the polyhedrin gene in the virus genome. During co-transfection both transfer vector and the virus genome are introduced into the host insect cell, in which the homologous recombination occurs and the gene is inserted into the parental virus genome. The genome then replicates to produce recombinant budded virus (BV), which can be harvested from the culture medium.


If unmodified virus genome is used, only about 0.1 – 1% of the produced virus is recombinant, and it can be separated from the parental virus by plaque-assay and plaque-purification.

Higher percentage of recombinant virus is obtained if the viral genome is linearized at a site located near the target site for insertion of the gene of interest (30%)1.

Even better results (over 90%) are obtained if the linearized genome is missing a portion of the essential gene downstream from the polyhedrin gene (ORF 1629)2. Such modified viral genome is sold under the name BacPAK6 and you can use it to produce your own recombinant virus.

BacPAK6


Figure 1. Map of the BacPAK6 vector showing restriction sites and orientation of genes


Site-Specific Transposition (bac-to-bac):

In this approach the gene of interest is cloned into pFastBAC vector which is then used to transform DH10Bac competent cells.  This particular strain of E. coli contains the bacmid with a mini-attTn7 target site. The mini Tn7 element on the pFastBAC transposes to the mini-attTn7 target site on the bacmid inserting the gene of insert and disrupting at the same time the LacZ gene. Recombinant colonies can then be selected, picked and amplified. DNA obtained from these selected cloned is used to transfect insect cells3.


References

  1. Kitts P. A., Ayres M. D., Possee R.D. (1990) Linearization of baculovirus DNA enhances the recovery of recombinant virus expression vectors. Nucleic Acids Res. 11(19), 5667-72.

  2. Kitts, P.A. and Possee, R.D. (1993) A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques 14, 810.

  3. Anderson D., Harris R., Polayes D., Ciccarone V., Donahue R., Gerard G., and Jessee J. (1996) New baculovirus expression vectors for the purification of recombinant proteins from insect cells. Focus 17, 53

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