I have a lentiviral vector that has a HSP70 promoter. My experiment relies on the low level expression of my gene of interest, too low that I normally can't detect it in western blot.
I would love to modify this vector with a reporter such as GFP, for example. I am just wondering if anyone has used such vector, and if the promoter is good enough to drive the expression of GFP to be visualised under flow cytometry or microscope.
The 2nd question is that I could rely on the leakiness of an inducible vector (Tet inducible but without Dox, either TetR or rtTA). I am just wondering what the level of gene expression is if leakiness were to occur, compared to that driven by HSP70 or UBC promoter. Anyone has any experience in this?
Many thanks!
Hi Milon what you could do is but in an IRES (internal ribosome entry site) into the vector i.e. HSP70 promoter-GFP-IRES-gene of interest.
The strong promoter drives the synthesis of the RNA that contains GFP and gene of interest. However translation of the gene of interest will be weaker from the IRES than the GFP. The GFP and gene of interest will not be fused in this construct.
Alternatively fuse the GFP and gene, transfect cells and select weakly glowing/faint cells for analysis/clonal expansion.
However both of these may be too strong still if you want almost undetectable expression.
Crazy idea - you could transfect a GFP fusion construct then hit it with siRNA/dsRNA against the GFP/gene. Silencing expression using RNA interference is generally never 100% effective (but >70% perhaps)? Not recommending this approach but just suggesting looking at the problem from a different angle?
Hope this helps...
I use a lentiviral vector with the EF1a promoter, which is weak. We use this promoter to transduce bone marrow cells, which will silence strong promoters like CMV. But the promoter for your gene of interest need not be the same as the GFP promoter! You can use a vector that has EF1a-yourgene-PGK-GFP (this is not a fusion, two separate proteins are produced). The PGK promoter will be plenty strong enough to see the GFP with a microscope. In my experience an IRES-driven GFP is very very weak. And be aware that in either case, the larger your gene is, the more the GFP brightness may be reduced.
If you can order things from Addgene, I recommend Addgene #17618 EF.PGK.GFP! I have cloned my (3.1kb) gene into it and the GFP looks great!
Hi Milon,
Maybe this is too late to answer you but I would like to suggest you to use a bidirectional promoter (PGK/miniCMV) which works well, furthermore you have a differential of expression which could fit with your need, indeed, PGK is a weak promoter and CMV mini is stronger.
Regards
Nicolas
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