Thread: Co transfection and gene reporter assay

Hi there,

I want to analyse the activity of a protein through expression vector (Luciferasse). So I select pGL4.30[luc2P/NFAT-RE/Hygro] Vector, and my question is, wich vector of renilla is the best for my experiment like a basal control? I mean, if the renilla vector must to have the same antibiotic? what things I must to consider?
I have the list of the vector that I could choose, but I want to have parameters to decide it.
http://www.promega.com/products/repo.../pgl4-vectors/


Thank you!

Kitty

what other control would I consider? maybe some without promotor?

The link does not work.

I don't know how you want to transfect? Do you use a plasmid, or phagemid, or even a m13phage. I usually use ampicillin, and an ampicillin resistence on my vector. I bet you want an E.coli strain that is resistant, but maybe missing a factor.

Picking parameters (i wasn't to good at this in school), start with if the bacteria will react on it, use selective markers the bacteria doesn't already have. And same with elective markers. Limited bacteria must be supplied with their missing link, before putting them on culture where they can not survive without this factor.

For the rest i don't have enough information to help you.

Hullo,

pGL4.30[luc2P/NFAT-RE/Hygro] contains an ampicillin resistance gene for selection in E. coli so my guess is that if you are planning on mini- or maxi-prepping this vector, then the basal control vector will need to contain the same ampicillin resistance gene. pGL4.30[luc2P/NFAT-RE/Hygro] also has a mammalian selectable marker for hygromycin resistance, so this may be worth considering if you plan on transfecting your vectors into mammalian cells - your basal control would also require a hygromycin resistance gene if you wish to select for transfection-positive mammalian cells using both vectors.

In terms of expression level, pGL4.30[luc2P/NFAT-RE/Hygro] is designed for over-expression of the protein of interest. As a basal control, it might be worth researching the endogenous expression levels of your protein of interest in the mammalian cells which you will be using experimentally. According to the product information, pGL4.30[luc2P/NFAT-RE/Hygro] is intended for use in HEK 293 cells. So, it might be worth finding out the basal endogenous levels of your protein of interest in HEK293 cells first, and then identifying the renilla vector that gives an expression level closest to the endogenous levels (so that you can compare the protein activity when over-expressed versus the protein activity when expressed at near-physiological levels). Also - is your protein of interest normally expressed in HEK293 cells? If not, you may wish to question the physiological relevance of any findings gained in this cell line.

Another control that you could use, is a parent vector (vehicle-only) control. I.e. transfect cells with the parent vector containing the gene encoding the protein of interest alongside the blank parent vector (not containing the gene encoding the protein of interest). This way, you will be able to observe any effects that may have arisen as an artifact of simply transfecting in the parent vector, independently of the protein of interest.

A final word - be careful in choosing the most appropriate vector - in order to ensure the reliability of the results gained and also because the vectors are quite expensive!

It would be well worth discussing this with your Supervisor.

Best wishes,

Tridimity