2 replies to this topic

[J]

I'm transiently transfected mammalian cells with my DNA constructs for reporter gene analysis.
As a control I co-transfect another vector to allow for variation in transfection efficiencies etc etc etc

Does anyone have an idea how likely it is that these reporter vectors are going into the same cell, baring in mind I only have an approximate 10% transfection efficiency?

If not, any ideas of a reference where the two reporters used for this purpose where visable by staining or some other method, perhaps B Gal and GFP?

[rs]

You know, I was about to ask exactly the same question myself!! It's an interesting theoretical question, however, for reporter gene analysis, I don't believe that it's at all neccessary for the second 'control' vector to enter the same cell as your 'test' vector. All you are attempting to do with the control vector is make small adjustments for transfection efficiency. Regardless of whether in the same cell or not, the concentration of the control reporter should reflect the transfection efficiency.
However, if you are performing co-transfection experiments where it is neccessary for both vectors to enter the same cell - for example if you are cotransfecting with a vector that encodes a transcription factor whos activity wrt your promoter you are investigating - how do you ensure this if you have only 10% efficiency? There are some vectors available that have two promoters for co-expression of two proteins in the same cell (Invitrogen) but I know of no others for reporter gene work.

Good luck!
Rachel

[milanoj]

If you are only interested in efficiencey co-transfection is not necessary. Whether both constructs enter the same cell is irrelevant. Your transfection efficiencey can be estimated by doing a side-by-side transfection. This will give you the info you want. What ever you do ignore the unnerving tendencey to express efficiencey in events/microgram. It is worthwhile to calculate about HOW MANY molecules you are attempting to introduce to about HOW MANY cells. If you really want to get fancey you can perform in situ PCR on your transfected cells for an accurate % of transfected cells. GOOD LUCK