So you have a transfer vector with some region homologous to the poxviral genome (to allow for recombination with the virus) but no poxviral promoter so you are thinking of trying CMV? Is that accurate?

The CMV promoter won't work, it is activated by cellular transcription factors, which are nuclear and thus not going to bind the poxviral genome which will stay in the cytoplasm during infection. The cellular polymerase will also obviously not be available in the cytosol.

The nice thing about poxviral promoters is that they are short and can be inserted upstream of a goi by PCR. Take a look at how we did it in Ibrahim, Jamin, Wicklund, and Wiebe 2013 in Virology. That is what I would suggest you do... which viral promoter you choose will depend on when you want the gene expressed.

@Matt

Thanks for the answer. Can I synthesize these short promoters as complementary oligos, with restriction sites, then anneal them for cloning?

That strategy can be used, as long as you take some care regarding the choice of restriction sequence at the 3' end of the promoter. Multiple studies have shown that placing 'extra' sequence between the pox promoter and the ATG can result in less efficient expression. papers Baldick et al in the early 90s and by Knutson and Broyles might be good to look at.

@Matt

Please allow me to contact you on your official email. I have landed into problems with the PCR incorporating the pox promoter.

Follow up on own question:

The purpose is to generate a GFP-expressing recombinant virus. A short (approx. 40 bp) poxvirus promoter is inserted into a plasmid upstream of a fusion protein (GFP::zeocin) and a poxvirus recombination site (approx. 500 bp) is inserted so as to disrupt the LacZ gene in the plasmid. The construct is then transfected into Vero, RAW264.7 or L929 cells using either Lipofectamin or Xfect (Clontech). Another batch of cells is first infected with ectromelia virus Moscow strain 2 h before transfection with the same plasmid. The same plasmid, but carrying the poxvirus homologous recombination site and GFP::zeocin placed under a CMV promoter is also transfected in yet another batch of the same cell lines.

Result: (1) 24 h onwards there is expression of GFP in all cell lines transfected with the plasmid carrying the poxvirus homologous recombination with GFP::zeocin under CMV promoter, (2) There is NO GFP expression in cells transfected with plasmid carrying GFP::zeocin under the short poxvirus promoter, (3) there is GFP expression in all cell lines previously infected with ectromelia virus and transfected with plasmid carrying GFP under poxvirus promoter, (4) first passage of viral progeny from result (3) does not show any GFP expression. We believe we do not have any GFP virus recombinants. Our minds are not prepared for this, so what’s happening?

Hello Felix,

Did you get this sorted out? The recombination frequency can be quite low, so on first passage you may not see much signal, and it may be very quickly competed out by wild-type virus.  To get the recombinant virus you would have to amplify it under drug selection. I don't have much experience with zeocin, but it is not the strongest selection system for poxviruses, and the drug is quite labile.  Blasticdin and G418 systems both work very well.

Hopes this helps (if you still need it).

Cheers,

David

Sirs: David Ulaeto and Felix Toka

I have another doubt in this.

If I constructed a recombinant plasmid in the following order of genes in pCIneo plasmid:

Fowlpox virus gene 1- early late promoter of poxviral origin - Non-poxviral gene - Fowlpox virus gene 2

Will CMV promoter be able to express the non-poxviral gene? Or the expression of the non-poxviral gene will be dependent upon the poxvirus promoter that was inserted?

Hello Ranjani, if you are putting the plasmid into a cell line, the poxvirus promoter will not be active. If there is no stop codon between fowlpox gene 1 and the non-pox gene, you may get a fusion protein of fowlpox gene 1 and the non-poxvirus gene from the CMV promoter, but only if they are in the same frame.

If you are using the plasmid to make a recombinant virus with the non-pox gene in between the two fowlpox genes in the virus genome, then the poxvirus promoter will drive expression of the non-pox gene in the recombinant virus. It will also drive expression in cells that you transiently transfect and then superinfect with a poxvirus - but not in a long-term stable cell line where the construct is only present in the nucleus.

If you are making a recombinant virus, you will need to check that you have not disrupted promoter or terminator sequences for fowlpox genes 1 and/or 2, as these non-coding control sequences are often embedded in the coding sequence of the adjacent gene and are easily disrupted by intergenic insertions.

Cheers,

David