2 replies to this topic

[gyma]

Hello everyone, I am making a lentiviral vector that expresses 1 gene of interests by CMV promoter and 1 GFP marker by hPGK promoter. I want to know will this kind of combination work? They are both pol II recruiting promoters so it is supposed to see competition between these two, right? But we have numerous genes expressed in our body at the same time, how is the competition of pol II in there? Is it a problem of distance? Usually two genes will be much more apart in our chromosome than in the vector, right?

In my case, if I do this kind of cloning, when the sequence is getting integrated, how is the expression like? I guess I will have two transcripts, is this correct? No more others? Where would CMV-induced transcription stop? at the end of GFP or only the first gene? It depends on the location of poly A signal, right? what about if I use IRES for GFP? I read it somewhere that IRES is only working for translation from middle of an mRNA. If this is true, if there is a poly A in the end of my gene to stop IRES-GFP transcription, the GFP expression will not exist anymore, right?

Sorry for so many questions. Hope some one can be patient and answer me. Thank you very much.

[pcrman]

If you want to compare the genes/promoters in a vector to genes/promoters on a chromosome, there are mechanisms that separate neighboring transcription units into independent ones such as the presence of insulator. In your situation, upstream promoter activity may leak into the next one even you have terminator in between. In you let your construct integrate into the genome, you may get one or two transcripts.

You should have a read of the following papers which address your questions well:

Two-promoter vector is highly efficient for overproduction of protein complexes
Lentiviral Vectors with Two Independent Internal Promoters Transfer High-Level Expression of Multiple Transgenes to Human Hematopoietic Stem-Progenitor Cells

[gyma]

pcrman, on 18 December 2012 - 11:03 PM, said:

If you want to compare the genes/promoters in a vector to genes/promoters on a chromosome, there are mechanisms that separate neighboring transcription units into independent ones such as the presence of insulator. In your situation, upstream promoter activity may leak into the next one even you have terminator in between. In you let your construct integrate into the genome, you may get one or two transcripts.

You should have a read of the following papers which address your questions well:

Two-promoter vector is highly efficient for overproduction of protein complexes
Lentiviral Vectors with Two Independent Internal Promoters Transfer High-Level Expression of Multiple Transgenes to Human Hematopoietic Stem-Progenitor Cells

Thank you so much, pcrman. I have read carefully the second paper you posted and it really helped me a lot. They came across the problem frequently that IRES-induced GFP/RFP doesnt express well whereas the upstream cassette is working well. This made the "marker" function meaningless. Finally they solved this problem by using two promoters for target gene and GFP/RFP independently, instead of using IRES.
This reminds me of another question that I posted in this thread recently. http://www.protocol-...iciency-at-all/
My observation is weird and similar to theirs. However, in my system, I have U6 for shRNA expression while CMV for GFP. It turned out too strong GFP expression resulted in no shRNA expression and I couldnt know why. Now I guess maybe for shRNA expression, a CMV promoter in the downstream may be too strong that it might disrupt the upstream function.
Anyway, now I can continue to make the vectors and test whether they really work. Thank you very much. I have learned so much here.