Antibiotic resistance-GFP fusions working? - Anybody with experience with pTracer and friends? (Dec/19/2007 )
Hi,
I am looking for a possibility to pack
a) my gene of interest
b) GFP/something colourful
c) a selection marker
together into one vector (ideally a retrovirus). I came across some vectors which express zeo (or whatever else resistance gene)-GFP fusion proteins like in Invitrogen's pTracer constructs.
Did anybody here ever try something like this and could please help me out? I'd like to know whether
a) this is actually working?
b) how long it is working (does the fluorescence disappear after a few days? or does it take much longer to come up than in a native GFP)?
c) is this something you liked working with and you'd recommend?
d) is it depending on the cell type you use (I'd like to use this for both primary cells and cell lines, both neural stem cells)
Comments are much appreciated....
Thanks a lot :)
Dedee
Do you really have to have an antibiotic resistance plus a GFP marker?
Can you do a fusion protein with your protein of interest and GFP?
Have you tried Clonetech vectors? They have an interesting range of colors with the ability to clone gene of interst as a N-terminal or C-termial fusion with the F protein.
Our lab uses some vectors from Invitrogen but then we also modified it for our needs. Some of these vectors have gene of interest, fluorescent protein and selection marker. I think most of the labs do use such vectors and I dont think its a problem. These are all lentiviral vectors.
One thing, to remember is if the promoter in these vectors will work in your cell line or primary cells or not.
In cell lines, we do see fluorescence in a day. Give it 2 days and its really bright.