B-galactosidase variability in luciferase assays

[estersan]

Hi,

 

I'm having problems in b-galactosidase stability during luciferase assays. Typically we transfect cells (different cell lines) with Lipo2000 in 24wp using 0.15ug pGL3 luciferase-reporter gene; 0.1 ug SV40-bgal (or 0,025 ug pCMV-bgal) plus 0,3ug of our different repressors of interest (some of them also under pCMV control). We made equal the final amount of DNA/well with pBSKTT gene. We transfect them during 4-6-12h and change the medium and analyze by luminometryafter 48h .

We observed good luciferase signals (low background, 200-300.000 RLU).

We measure b-gal adding corresponding substrates (sometimes the half of the indicated reactive) and after 1h of incubation.

About b-gal... measures indicate high background signal (40.000) and low transfection (similar to background-although luciferase doesn't indicates this!) or good signals (300.000) in comparison to background but with great fluctuation (from 20.000-1x10^6). My boss thinks they should mantain constant.

 

So, what's happening? We are questioning everything! DNA quality seems to be good (A260/280=1.8) although differences in bacteria transformation have been seen depending on which plasmid you're growing. Is b-gal a good indicator of transfection efficiency or is it also modulated? Low backgrounds are typically solved doing 1h at 50ºC. However, when we did it, everything was "dead". We have also compared different kits (Clontech, Roche, Tropics) and although the signal changes the proportion is mantained. Why does b-gal change depending on the cotransfected plasmid? Looking at the bibliography some people offer alternative techniques such as Slot blot, PCR...which involve quite a lot of work for a periodical technique.

We are also thinking about using Renilla as measure of transfection efficiency; although some workmate has also observed variations in presence of TGF-b.

 

It's kind of a common technique, and any kind of help about this topic would be really appreciated!!

Thanks a lot!

[CharonY]

Is b-gal a good indicator of transfection efficiency or is it also modulated?

Normally beta-galactosidase is fairly stable, hover the viral promoter (in this case CMVp is sometimes not expressed stable and reliable in all types of cells).

Maybe this can be a factor.

 

Why does b-gal change depending on the cotransfected plasmid?

OK I did not quite get that one. Did you observe a change of beta-galactosidase levels if you co-transfect it with a different plasmid than pGL3?

 

Another effect might be dilution. AFAIK the luciferase gene in pGL3 is under the controle of the SV40 promoter. If the beta-galactosidase is, too, then you might get competitive effects.

[estersan]

Hi,

 

thank you very much for your reply.

 

Yes, we have observed that depending on the cell type (we have tried MCF-7, Saos2, U2OS, C33A, SW13, C2C12, etc...). In fact, there are several articles that explain that it's not realiable to compare promoters in different cell lines.

 

About cotransfected plasmid: we observed that CMV-Galactosidase increases when we cotransfected a pBJ5-gene or pBA-gene2 or even with pBJ5-gene2. Independently of Luc values. We typically use Luc, b-gal and transcription factors under different promoters in other to avoid competition of transcription factors.

 

Thanks again!

[CharonY]

Oh, I think it got you now. So you are saying that the beta-galactosidase levels change if the vector of the carrying the regulator, or even if only the regulator changes?

 

Hmm is it possible that the repressors in question have a weak affinity to the promoter of the beta-galactosidase?

[estersan]

Yes, b-gal changes mainly if the regulator changes. I would suppose that CMV require constitutive promoters that are not interfering with the TF- carrier promoters. Curiously we have observed this effect as well using SV-40 promoters.

Somebody told me that he has observed also variation in CMV using p53 transcription factor; and there is an article that states that, at least doing calcium phosphate transfection, there is transient G1 stop that induces p53.

As solutions with problems in b-gal variations, I've read about introducing directly the b-gal protein, so promoter-independent measure of transfection.

 

I'm thinking about trying the minimal RSV promoter and Renilla.

 

Thanks for the brainstorming!

[CharonY]

Introducing proteins directly sounds interesting. However I am not sure if it is a good measure of DNA transfection. But then I assume that the authors will have made comparisons. In any case, I would be interested to hear what worked for you.