complicated transfection requires professional help - (May/24/2009 )
I intend to use the dual-luciferase reporter assay for quantitative measurement of gene expression.
I want to produce stably transfected cells that will express both renilla and firefly luciferases under the control of independent promoters and that the cells will also be expressing GFP constitutively (necessary for sorting the cells by FACS).
I badly need help to address two problems:
1) I need an internal control to relate the luciferase expression to that allows stable expression assay (i.e, contains selectable marker and is constitutively expressed)
2) I guess that the excitation of GFP by renilla luciferase might be a problem as the light emitted by renilla reaction is at 480 nm well within the peak for excitation of GFP (470-490 nm). will that actually happen?
Any advice will be highly appreciated
Thanks in advance
GFP will be excited by the light generated from the renilla reaction, but it may not be detectable as there are usually significant losses in fluorescence. You will need to run a control for this experiment using non-transfected cells, transfected cells that produce the GFP alone, cells that express the renilla luciferase... etc., I am sure you can work out the permutations.
many thanks bob1 for your reply but what about the 1st question. is there any company that provides vectors like B-gal that can work as an internal control BUT CONTAINS A MAMMALIAN SELECTABLE MARKER? because in my case i am not going to use renilla as an internal control
Try looking here:'http://plasmid.med.harvard.edu/PLASMID/Home.jsp