Hi Huanyao,

Well, there are, as far as I know, at least two "pEYFP-Y1" constructs around ... Both are expressing enhanced yellow fluorescent protein (EYFP) but the Y1 behind is different in the two constructs: in one case, Y1 stands for neuropeptide Y1 receptor, in the second case it stands for Y1 version of EYFP (the Venus superenhanced version) ... 

In both cases, the initial vector was pEGFP-N1 from Clontech. I attached a pdf file quickly describing this initial vector. I'm afraid there is no "backbone" vector available.

Best regards,

Philippe Urban

Are you trying to optimise the transfection? If that is the case then by using a constant total amount of DNA you will have the same efficiency every time, just with less or more yellow going on, proportionally. The whole point of the optimisation is to vary the quantity of DNA, (and/or the ratio of DNA to transfection reagent, depending on the reagent). Or do I not understand your question?

Thank you all for the answers. 

And yes, I'm trying to optimize the transfection condition in order to minimize the cytotoxicity, caused by overexpressing my protein of interest, which is fused with YFP.

I'd like to maintain the total DNA and total DNA/transfection reagent ratio both the same, but just vary the amount of my YFP DNA while supplementing with more empty vector. I'm considering using very simple empty vectors such as PUC19 or PBR322, but not sure if they are the right one to go to.

I see, if your protein of interest is toxic I can understand why you're doing that. But keep in mind that there can be toxicity from the transfection reagent (which is cell line dependant) and the total mass of DNA you're putting in there. You can minimise this by washing the transfection reagent off after 6 - 8 hours, rather than leaving it on overnight. Honestly, I wouldn't control for total DNA content, I would just optimise the transfection to give minimal toxicity while generating a workable signal. After all, you wouldn't perform your actual experiments while putting junk DNA in there.

Transfection efficiency  is a complex issue,  your input amount of DNA, your particular ways of preparation for the plasmid, your transfection reagents, your cell type of interest and your particular DNA construct, the growth status of your cells, the particular promoter of your constructs some times a particular media for the cells etc all play roles towards your final read out of the efficiency.

A positive plasmid (such as pEGFP-N1) and a non plasmid control (transfection reagent only)  will give you ideas of how your efficiency, toxicity,  of the transfection to your particular cell types. You can dilute your plasmid of interest or a positive control plasmid to a few different concentrations, and titrate the plasmid using 100ng, 500ng, 1ug, 2 ug etc for your tests. Please keep in mind, the particular ways of DNA preparations will affect your transfection efficiency.

If your backbone control is for the auto fluorescence concerns, you may including any non reporter DNA. However, auto fluorescence  can be caused by other reasons more than DNA itself.

They will be auto fluorescence from the transfection reagents, and most of the transfection reagent will have some toxicity, which will lead cells to die therefore again resulting in secondary auto fluorescent.Over production of the fluorescent protein by the strong promoter such as CMV etc will  also have toxic affect to various cell types resulting cell death.

A negative control of the backbone plasmid may or may not needed in the experimental settings unless you think there might be leaking issues of particular form of the reporter constructs you use or mutations etc

Thank you all very much for your answers.

I think it's a good idea to try DNA-free sample and decrease the amount of total DNA (and reagent) to at least have an idea about the toxicity caused by either overexpressing my protein or the reagent itself. 

Hi Huanyao, you can try other transfection reagents such as Magnetofection or DreamFect Gold, depending on your cells.

http://www.ozbiosciences.com/content/13-magnetofection

http://www.ozbiosciences.com/cell-lines/19-dreamfect-gold.html

If you need samples let us know at [email protected]

Good Luck

Olivier