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I have two proteins and I will be preparing a vector with both genes for stable transfection. Each protein will have their own promoter and I will use piggyBac vector to insert a single cassette with both genes in it into the chromosomes. My questions are:

  1. If I use the same promoter (CMV, SV40 or an inducible one) for both genes how would this affect the expression levels? I read in some papers that one of the genes might get expressed lower than the other. If you have first-hand experience I will be grateful if you share it.
  2. If I use the same promoter for both, but put them far away (i.e. ends of the cassette) would the expression levels get affected again? It sounds simple enough but I think people would have published something if they had a good result with that.
  3. If I use different promoters for each gene (e.g. CMV for one and Tet-inducible for the other) how would it work? I couldn't find a paper which used this and I would like to continue with this approach; therefore, if you have any experience I will be really grateful for any help.

I don't want to use IRES or 2A-peptide. I would like to be able to control this experiment in transcription stage.

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You can use a bidirectional promoter. The problem that you mentioned about proteins not expressed in same level happens because of competition for polymerase. But there are well optimized parts and also commercially available vectors that work fine.

You can clone genes in a serial order. It won't be a problem. Just leave a 100bp linker after the polyA signal of previous gene. If your cassette becomes too huge, then insertion becomes slightly difficult. That's why people use IRES or TA-peptides, etc.

Retroviral based insertions work quite decently, but you can't control copy number variation between different cells.

You can use two different promoters. The dynamics will depend on the promoter strength and concentration of inducer.

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  • $\begingroup$ Thanks for your reply. I looked into the bidirectional promoters too but decided to try two different promoters. I really didn't want to go with IRES or 2A because I am too afraid to interfere with the interaction, better not risk it :). I will start the experiment shortly and share my experience in here when I get some results. $\endgroup$
    – Engin Yapici
    Commented Apr 9, 2013 at 22:28
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I was going through my questions and realized this is still unanswered. Well, I have the answer now.

I designed the construct with two separate promoters (CMV for one and Tet-inducible for the other) and got really good results. I haven't had any difficulties in expressing the proteins. I was able to control the Tet-inducible one very nicely and got steady levels of the other protein with CMV.

Long story short: you can safely put two genes under two separate promoters, prepare stable cell lines with them and get good expression levels.

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