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Co-transfection of multiple plasmids


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#1 DrSnood



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Posted 29 July 2016 - 05:44 AM

Hi All,


I'm trying to perform a reporter assay using multiple plasmids. In short, I want to see if a combination of TFs activates my reporter. I have my system working well with one plasmid and my positive control reporter. However, when I express all 7 plasmids (Renilla, firefly, and 5 TF expression constructs) required for the experiment, my positive control no longer works, and I'm unable to detect expression of my transfected plasmids by western. I am using the same amount of DNA for my positive control (bringing up total DNA concentration with empty vector) and the experimental transfection. ANy suggestions on how to begin optimizing this system? Is the expression of 7 plasmids even realistic (I'm using U2OS cells)? Thanks!



#2 827753



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Posted 02 August 2016 - 08:40 PM

Can you get 7 distinct fluorescent reporters and transfect 1, 2, 3, 4, 5, 6, 7 to see where efficiency begins to drop? (Or even just do this with the TF plasmids and your positive control?)


Are these all using the same promoter?  Perhaps some gene silencing is happening?


Could you tandem some of the TFs onto a single plasmid with a single promoter via a 2A peptide "fusion" to reduce the number of plasmids?


Back when I was an assistant working on eukaryotes I don't think we ever went in with more than two plasmids, so really can't help, but hope these questions might help.


Good luck.


P.S. What's the transfection efficiency of the positive control?  Is it enough that it seems likely a noticeable number of cells would uptake all 7 plasmids?

Edited by 827753, 02 August 2016 - 08:47 PM.

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