Dual Luciferase Assayproblem with cotransfection of reporters - (May/08/2006 )

Hi,

I have been trying to transfect my cells with Luciferase (PGL 4) under control of p53 and Renilla under control of TK (phRL-TK) for transient transfections. However, when i transfect them together, the values increase enormously comapred to when they are separately transfected. I don't think this is due to interference of the luc substrate into the renilla substrates as both luc and renilla activity increase. How can I overcome this? My ultimate ain is to use these vectors for stable cell lines.

Would really appreciate some ideas..thanks!!

Molbionovice.

When you say 'under the control of p53' what exactly do you mean? Do you mean under thr control of the p53 gene promoter?

Do you have different transfection conditions in each case. How much DNA are you adding and how much transfection agent are you using? Are you incubating for the same period of time?

Have you checked that your transfection efficiency is the same in both cases?

If you can put the exact protocols for the two situations on the forum it might give more insight. It seems odd that it would be an interaction. Are you treating your cells with anything?

Taking into consideration exactly what you r saying:

It seems that you might be overloading your cells with a lot of negative charges when you are transfecting both the plasmids together. You need an optimal Nitrogen to Phosphate ratio (N/P ratio) for the DNA to permeate the cell membrane.

Increase the amount of transfection agent when you r adding both the plasmids and you shold be fine..

Hi
Thanks so much for your replies.


1. Yes, I mean that the luciferase is under the control of the p53 gene promoter. This is actually 13 binding sites of p53 which are concatemerised.
2. More details of my experiment- I did a series of 5 ug. 0.5 ug and 0.05 ug of LUC each with 1 ug GFP ; 5, 0.5, 0.05 ug of RN each with 1 ug GFP and 5 ug of LUC and 0.5 ug RN together with 1 ug GFP. The DNA conc is made up to 6 ug with TOPO vector. The RLU for 5 ug of LUC only was 5092, 0.5 ug RN only was 327 and when together, LUC was 20149 and RN was 728. These are values normalised to GFP transfections (given below).

Transfection efficiencies (in %) were 36, 31 and 13 for 5 ug, 0.5 ug and 0.05 ug LUC; 10,15 and 3 for 5 ug, 0.5 ug and .05 ug RN and 13 for 5 ug LUC and 0.5 ug RN together.

3. I am not treating my cells with anything.

I am transfecting a total of 6 ug DNA into my cells (I plate 2.5 million cells on a 10 cm dish the prev day and add this DNA to it). I am using the FuGene Transfection reagent and adding 36 ul to eahc tubes (6:1 ratio of Transfection Reagent:DNA).

Hope this helps

Molbionovice

Nope, I've no idea what's going on there then! Sorry.


Just one point; the 'p53 gene promoter' means the promoter which controls the expression and transcription of p53 mRNA, NOT any promoter to which p53 binds and exerts an effect.

That said, I do believe that p53 does, in fact, bind and regulate its own promoter. From what you said you have thirteen contiguous p53 binding sites in your promoter. That sounds like a synthetic promoter to me not the p53 gene promoter.

Yes, it is a synthetic promoter. It has been used in a number of other studies and also in a luciferase assay in HCT-116 colon cancer cells along with RN expressed from a retrovirus. How can I find more info about the Nitrogen/Phosphate ratio? Any ideas?

Thanks!..