transfection of HiFive Cells? - Baculovirus Expression (Sep/06/2005 )

Hello,

I am in the early stages of expressing a protein using baculovirus. My problem is that I can not seem to prevent fungal contamination of thawed Sf9 stocks. I do add Fungizone and Penstrep to my media, but this does not seem to be helping. I have had difficulty pinning down the source of the contamination as it appears to potentially be originating from several sources (perhaps the shaker and cell stocks). Unfortunately I only have one source of Sf9 cells and these may be a source of contamination. I was wondering if anyone could comment on the possibility of transfecting Hi5 cells directly rather that first transfecting Sf9 cells and then expressing in HiFive cells. We have a source of HiFive cells that appear to be growing up just fine without contamination.

In addition, since our shaker may be a source of contamination would doing the work in tissue culture flasks without shaking affect my yield dramatically. I am using the Invitrogen Bac-to-Bac kit.

Thank you for your help!

Just order a new vial of Sf9. Usually, recombinant baculovirus was generated from Sf9, then use the virus to infect Hi5 to over-express your protein. I don't think your bac vector can work in Hi5 directly. I'm not hearing anyone doing that.