We have a sf9 insect cell culture and until recently, we used the BD BaculoGold™ Baculovirus Expression System (BD Biosciences) for virus generation and it worked very well. Half a year ago the production of this system was stopped and we searched for another system compatible with the insect vector pVL1393 and sf9 cells. We found ProEasy (AB Vector) for the generation of recombinant baculoviruses. We tried it three times but it did not work. We have no idea why. Did anyone use the same system and has an idea what the problem could be?
Hi Deborah,
I have not used ProEasy baculovirus DNA, so can't comment on it directly, but I have successfully used flashBAC from Oxford Expression Technologies (OET) if you wish to try another source.
I'm assuming that you have run a control with a DNA transfer vector that you have previously recombined with Baculogold DNA, so that you know that there isn't anything wrong with your transfer vector DNA.
I'm also assuming that you have checked that you have no recombinant virus at all, using a plaque assay or by adding a high volume of S/N from the transfection to fresh insect cells to check for signs of infection - cell swelling, cessation of doubling etc. Sometimes with a very poor recombination it can be hard to tell apart transfected cells from the untransfected control as there is a large population of uninfected cells growing on that can mask the infected population.
Richard
Hello Deborah,
I am using the Bac-to-Bac-System from Invitrogen. In this system, the recombinant vector pFastBac1 is not co-transfected with linearizes virus-DNA and Sf9 cells. Before, transfection, recombination in Bacmid-containing E.coli cells is done. Perhaps, the co-transfection is the problem in your system because of the small positive result. Another way could be to lower the number of Sf9 cells during transfection as it worked quite good for me. If this does not work and your transfer vector is okay, I think Richard's idea of changing the system is a good solution.
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