Cloning gene behind GFP - Vector? (Feb/22/2006 )
I would like to clone the gene I`m working with behind a T7 promotor and GFP (to check for expression in mammalian cells). I found the vector pcDNA3.1/NT-GFP (Invitrogen) which would be just perfect. Unfortunately, I doubt I will be allowed to order it (472 €). Could I clone both GFP and the gene into the multiple cloning site of pcDNA3.1 instead? If so, how?
I normally use pcDNA3.1 without GFP which forces me to check the expression by doing a Western Blot (problems here: low transfection rates, antibodies not easily available, no positive control).
You can indeed clone both your EGFP and your gene of interest into pcDNA3.1. Only thing you probably want to make sure of is that you're cloning in frame or add an IRES or so. Apart from this, clontech has a lot of vectors for cloning genes, but there not working with EGFP anymore, but with new (according to them of course better) fluorescent proteins, AcGFP, ZsGreen and so on. go to www.clontech.com for more information. There are also some other companies selling GFP-variants like promega, stratagene, evrogen (www.promega.com, www.stratagene.com, www.evrogen.com) and several others...
Btw: T7 promotor is a bacteriophage promotor, so in eukaryotic cells (as you mention transfection I assyme you're working on a cell line) it won't induce too much expression. But maybe this pcDNA3.1/NT-GFP also contains CMV (cytomegalovirus promotor which is a strong promotor) like the other pcDNA3.1's?
Yes, I guess there is a CMV promoter. What kind of IRES site would I have to use to express EGFP or my gene (filarial) in cos7 and HeLa cells? Is the detection of GFP fluorescence in these cells after transfection then a proof of expression of the filarial gene as well? We normally use GFP as a positive control in transfection to get to know the transfection efficiency.