Hi Laura, have you tried centricon concentration filters on the virus? 

no- what size?

Probably YM-100 will be the fastest way to concentrate the virus.

http://kirschner.med.harvard.edu/files/protocols/Millipore_Centricons.pdf

Hi, Laura,

we are using insect cells expression for long time and we never filtered the viruses. My viruses last for 1-2 years at 4°C and in dark. We use Gibco media for S9 and Expressions media for SF21 and Hi5 n(ESF 921 Insect Cell Culture Medium, Protein-Free96-001). The last gives very nice expression and the cells are happier than in Gibco for Hi5. And the media cost only 36 euro per L.

I would start with new preparation of the virus. Do you have some control on your bacmid like YFP or other marker to see if the production of V0 was ok? We are using protocols from Imre Berger. When producing V0 I do incubation only for 48-60 hours, than spin down at 3000rpm/3min. The supernatant I store at 4°C. You can store it also for longer time. Don't use too much for infecting for V1. Important is that  first day after infection the cells have to doubled.

I hope it will help.

Curr Protoc Protein Sci. 2008 Feb;Chapter 5:Unit 5.20. doi: 10.1002/0471140864.ps0520s51.

MultiBac: multigene baculovirus-based eukaryotic protein complex production.

Bieniossek C1, Richmond TJ, Berger I.

.

HI Laura,

A couple of things to look into:

How does the growth curve and viability look after infection to generate your P2? If your starting virus is a very low titre then the cells will outgrow the virus generation and you will get a low titre. Normally cells infected at MOI≈0.1 and 1.5e6/ml will grow to ≈3e6/ml and then plateau, but if they keep growing it means your starting MOI was much lower. Inoculation with more virus can overcome this.

Another possibility is that there is a 'toxic' effect from expression of the protein, if so you will constantly get Titres around 1e6 pfu/ml from a correct MOI. Unless there is some way to stop this 'toxicity' then there's not a lot you can do to improve the titre. You can still use the virus but you might not see good expression in any case.

-Richard

Thanks to all for the tips, they are great.

It seems that our issue was in generating the bacmid itself.  We were using zyppy kits for bacmid purification, which apparently are not great for large DNA like bacmids.  We've since switched to the Purelink kits recommended by thermo and that seems to have helped quite a bit.

http://www.thermofisher.com/order/catalog/en/US/adirect/lt?cmd=catDisplayStyle&catKey=101&filterDispName=PureLink%26trade%3B+HiPure+Plasmid+Kits&filterType=1&OP=filter&filter=ft_1101%2Ff_172501*&_bcs_=H4sIAAAAAAAAAH2MQQqAMAwEX5OTKFpBvHrxLPiEGqlgjaSp%2Fb7rB4RlB2Zh647acVHZsrdUkRuq%0AlfU5PKcfH8xu6idyM1JKaSywRtmPBDZeInROKL5QQSIDohvGYPHEA7n%2BSzuaZgZedPbeBIcAAAA%3D

I have a question about the virus stability. After I generate my P1 or P2 virus stocks, I always keep them at 4C. However, after 2-3 days, they lose their infectivity. Even if I try really concentrate infections, they don't work. When I check the cell pellet, I see a lot of protein expression. The only weird thing about my virus production is that after addition of the virus, my cells stop dividing immediately (no first doubling). Can this be the source of my problem? Thanks a lot.

We've recently found that all reamplifications should be harvested on day 3.  Letting them go longer causes more cells to dye and the virus can bind to the dead cells and cell parts that are floating around. Then when it comes time to centrifuge your cells out, you are also losing a large portion of virus that is stuck in the cell paste.  Doing a day 3 harvest on all reamps has greatly increased our titer numbers and the stability of our virus.