Can I trust GFP-cotranfection - transfection control (May/20/2008 )
If I co-transfect cells with GFP-plasmid and a plasmid containing the sequence I am interesting in, can I trust that the cells that becomes green also contains "my" sequence? It is a large sequence (7200bp).
Thanks!
Most likely yes, particularly if you use a mixture of plasmid DNA with greater than 1:1 molar ratios of your construct vs EGFP construct.
Hmm
I would be careful about that.. If a cell has been succesfully transfected with the GFP plasmid, there are not guaranty it had been by your sequence as well. The better way to be absolutely sure your cell is transfected with your sequence of interest, is to put both on the same plasmid.
I think this probably depends on your transfection efficiency.
Say you have 2 plasmids, and that the chance of having a cell being transfected with both plasmids is the chance of it being transfected with one multiplied by the chance of it being transfected by the other (assuming that succesfull transfection with either plasmid is independent of succesfull transfection with the other).
If both plasmids would transfected 90% of your cells, then 0,9x0,9=0,81=81% of your cells would contain both plasmids, which means 90% of your EGFP+ cells would contain also the other.
In case your transfection efficiency would be 10%, then you would get: 0,1*0,1=0,01 or only 1% of your total cells would contain both, which means only 10% (1%/10%) of your EGFP+ would also contain the other.
I know that this is probably an oversimplification of things, and the mathematics would be more complicated than my simple example, but I would agree with Madrius suggestion. Having EGFP on the same plasmid would't make it drastically bigger (less than 1 kb for the protein, maybe you need an IRES or some other extra sequences, but relative to the 7,2 kb plasmid (or is that just your sequence? what's the size of your plasmid?) it shouldn't increase the size too much, and you can transfect efficiently with plasmids bigger than 10 kb in severall cell lines).
As a general rule, yes.
You can not, however, study one cell, and come to any valid conclusions. But if you study 100 cells, you can reasolably argue that most of the cells that contained GFP should have also contained your gene.
The mol ratio should be at least one, the odds improve if the ratio of "your gene"/GFP is greater than 1. And that should take into account any difference in teh size of the plasmids.
If you have to focus on a few cells only, you need to demonstrate by GFP + "Immunocytochemistry for your gene" that they were co-expressed in teh same cells.
Having both genes on the same plasmid also does not make it sure. It is quite possible to have GFP expression without your gene being expressed/expressed in sufficient amount.
You are right. One should also take into account the overall transfection efficiency. However, the cells that have been receptive to one plasmid, tend to be more receptive to two plasmids than the cells that have not been receptive at all. So, it is not really amenable to a mathematical formula.
You are right. One should also take into account the overall transfection efficiency. However, the cells that have been receptive to one plasmid, tend to be more receptive to two plasmids than the cells that have not been receptive at all. So, it is not really amenable to a mathematical formula.
Yes, that is what I have read to, that the two plasmid-DNAs form complexes with eachother and therefore go into the cells together.
My plasmid is about 12kb inkl "my" sequence.
How would you make that construct? Keep the promotor for GFP and have it as two separate units on the same plasmid, or have GFP after the same promotor as my sequence and use an IRES for it?
Why would plasmid A (containing EGFP) be more prone to make a complex with plasmid B (containing gene of interest) than with another copy of plasmid A?
(I do agree however that I indeed made an oversimplification of things, and that cells that take up one are more likely to take up 2, making taking up plasmids not "independent events").
Transfecting with a high molar ratio of plasmid with gene of interest verses egfp-plasmid would probably be easiest bet.
For making the combined plasmid: if you can (and are sure the 2 promotors work in your cell), you better have 2 different promotor (IRES would do the trick, but isn't as good as an independent promotor I believe and I don't know what effect 2 copies of one promotor would do).
Transfection process introduces complexes of DNA/reagent of up to million copies of plasmid DNA per cell, with no specific preferance at the dissociation/release and nuclear entery events. I am quite confortable at saying you have a very good chance of getting both plasmids in the same cell.