no GFP in cells - (Dec/04/2007 )

I made a clone with target gene at the N terminal of GFP expression vector. I delete the stop code of my target gene and design the primers so that the expression gene reading frame fiting to GFP expression reading frame. After sequencing, I get the correct one. But I cannot find GFP after I transfected it into mammary cells. note the GFP expression promoter is CMV.

What is the problem? how can I check it out?

Thanks

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And your positive control, the original EGFP vector transfected cells showed good transfection?

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yes, GFP control show good signals

I decide to make digestion to identified it again by 3 different ways

and I may send out for sequencing again

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Do you have the Kozak sequence in front of your gene?

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I had the same problem before with fusion proteins...the problem seems to be that some fusion protein interfere with the correct folding of the EGFP (it is a big barrel structure) and it can't fold proberly, the result is no fluorescence...in my experience sometimes putting a linker between the 2 proteins works better (4-5 aminoacids). Then they can fold more independantly. Or you could maybe prepare a construct with CMV-your gene-internal ribosome entry site (IRES)-EGFP instead of the fúsion protein

LG Stardust

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THanks. You meant that my protein may affect the folding of GFP right? I really did not think that way. But I use BamH1 as C-terminal of my restrict site on my target gene, there are 7 amino acids after the insert protein (Arg-Asp-Pro-Pro-Val-Ala-Thr-), then Met of GFP. Do you think 7 aa are enough to avoid the folding problem?

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we usually had few glycines between the 2 genes. Its supposed to be easier for the fluorescent protien to move around for proper folding.

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Is your protein expressed in these cells in general? What are the cells you are transfecting? Can you use different cells (like CHO, 293, etc)?

If your protein is not expressed/folded properly, there will also be no expression of GFP.

You can check for this either with an N-terminal GFP tag (for fluorescent expression) or with N-terminal tags like HIS or flag for western and check the correct size and the amount of expression. Idealy it would be if you can express the native protein and look for actvity. If you cannot see expression with these methods, your protein is not properly expressed and consequently also not the GFP.

In some rare cases esp in cells of the immune system, I have seen plasmid remodelling in a way that the GFP (or any other second protein of a fusion protein) was deleted from the plasmid. You could check for this by PCR after transfection and isolation of clones.

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