Adding CMV promoter to retroviral vector.

[mchlbrmn]

I have a version of the retroviral vector pMSCVpuro that has contains a 5'LTR, packaging signal, MCS, PKG promoter, Puromycin, and then the 3'LTR. To increase the expression, I wanted to add a 600bp CMV promoter just before the MCS. I'm not sure how this is going to work for expression with the two promoters? Any opinions? The longest ORFs in the CMV are 86 and 130 ish bp with imperfect Kozak sequences (although this probably isn't the major problem). Any opinions would be welcome. Thanks.

[CrowSan]

Hi I don't think I have ever come across a single gene with two promoters in the vector before therefore not sure how much any comments I make would make sense. However is it possible that having two promoters in close proximity might mean that proteins binding one promoter might block the binding of the second promoter pomoter (if that makes sense)?

There is an interesting article in PLosOne comparing promoter strengths:
http://www.plosone.org/article/info:doi ... ne.0010611
and the pgk promoter is fairly weak. Perhaps it would be safer (?) just to swap out the pgk for another promoter (e.g. CMV).

However saying that the paper above suggests that CMV was one of the most variable (cell type to cell type) and we have had a frustrating time with this promoter inactivating (almost certainly due to methylation) in H9C2 muscle precursor cells over time (this has been reported before - although we discovered the article after we had problems).

Another article (http://www.ncbi.nlm.nih.gov/pubmed/17609656) suggests that some promoters may act in a cell-stage specific fashion so if transfecting stem cells maybe the two promoter system may work better (?).

Anyway let us know what the expression is like (if you do compare single promoters to two together it may make a nice little paper!). Good luck!
CrSn

[mchlbrmn]

The viral 5'LTR is the promoter intended to drive the expression of the insert, but I wanted to leave the LTRs alone so that in the future it could still function as a retroviral vector. I was just going to try inserting a CMV promoter before the MCS to increase the expression level and see what happens. I appreciate the help, thanks.

[relaxin]

Vectors such as pLVX-IRES-ZsGreen1 (for map click link below) do have CMV promoter upstream from the MCS. You may have to add a termination sequence upstream from the CMV promoter to stop upstream transcription.

http://www.takara.co.kr/file/manual/pdf/PT4064-5.pdf

[mchlbrmn]

It's nice to see the two promoters in that vector, thanks. I'm not using a lentiviral vector, so I don't know that the ppt terminator is a good idea for me, but I should check more. I don't think I have any really usable ORFs upstream of my insertion site, so I hope premature transcription is not a problem.

I looked at the other two links, and they have information that may help out with other expression problems in the lab, where we had genes transfected successfully, but not expressing. The Pgk promoter in my vector is for the puromycin. The 5' LTR is the intended promoter, and after it I put CMV. I think I'll see what happens. Two promters may be better, if it's not worse, lol. (Well, actually I'm shooting for somewhat better).