I want to use BacMam to transfect mammalian cells, but I need to make the virus in insect cells. Should I leave the polyhedrin promoter untouched when I add a mammalian promoter to FastBac (baculovirus plasmid), or should I replace the polyhedrin promoter for the mammalian promoter (CMV)? Will removing the polyhedrin promoter have an effect in the expression and formation of the Baculovirus in Sf9 cells?
Fabian,
I don't think polyhedrin promoter matters for BacMam system. Your Sf9 cells should generate virus just fine without it.
Have a look at this article:
Dukkipati A, Park HH, Waghray D, Fischer S, Garcia KC "BacMam system for high-level expression of recombinant soluble and membrane glycoproteins for structural studies" Protein Expr Purif (2008).
The pVlad6 vector described in the article is available from Addgene for reasonable price:
https://www.addgene.org/41850/
Vitali
polyhedrin promoter here is for your gene expression. The virus harbours its own polyhedrin with its promoter. To replace polyhedrin promoter for the gene you are working with the mammalian promoter (CMV) is the right strategy for your purpose.
Sorry I am late on seeing this but Rick Boyce should be able to provide you with the pHTBV1.1 or newer BacMam vector. You would have to complete a MTA with Mass General. His e-mail is [email protected]
Molly :)
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