It completely depends on the fusion mRNA. But my gut feeling is that it is unlikely your GFP start codon would be used. Try the western blot as suggested above (Abcam #ab290 works well), but be warned that it is very likely you will find an unfused GFP band by western blot at high protein amounts and long exposures -- especially if you transfect your cloning plasmid. The reason is...

...although one ribosomal subunit accesses the mRNA from the 5' end, translation initiation can occur at any AUG (in any frame) along the length of the mRNA. That said, 5' AUGs are more frequently utilized than AUGs farther 3' (due to the higher frequency of Kozak sequences toward the 5' end and also scanning across other AUGs after initiating translation upstream; an exception to this is translation reinitiation where a ribsome starts at a second AUG after termination). In many instances, usage of downstream start codons is correlated to copy number of mRNAs, suggesting it's a probability game.

Alex Kochetov has done some nice work on this in recent years.

All this to say: In future projects I would think it is probably better to get rid of the GFP start codon so it will only be expressed as a fusion and you won't have to worry about alternative translation initiation mechanisms. Just be as careful as you can with the mouse you've already made. Maybe try localizing the fusion protein by IHC, though I'm guessing that's challenging and is why you made the GFP fusion in the first place.

Best of luck. Shoot me an email at [email protected] if I can be of any help.

Jonathan

It probably also depends partly if the vector also has a Kozak sequence that was left in. Empirical evidence is always the best though. Do a Western blot using anti-GFP? If you only have a band at the size of your protein+EGFP, and don't have a band at ~ 30-33 kDa (the size of EGFP if I recall correctly), then you are OK.

having the Start codon for both genes should not be a problem at all. Keep them in....

I don't think you will have this problem. In eukaryotes, translation is initiated at the 5' end of the mRNA molecule, since 5'cap recognition is required for the assembly of the initiation complex.

In order to initiate translation in the middle of your mRNA it would be required an "internal ribosome entry site" (IRES)

But, as said above, you can check that

Good luck

It completely depends on the fusion mRNA. But my gut feeling is that it is unlikely your GFP start codon would be used. Try the western blot as suggested above (Abcam #ab290 works well), but be warned that it is very likely you will find an unfused GFP band by western blot at high protein amounts and long exposures -- especially if you transfect your cloning plasmid. The reason is...

...although one ribosomal subunit accesses the mRNA from the 5' end, translation initiation can occur at any AUG (in any frame) along the length of the mRNA. That said, 5' AUGs are more frequently utilized than AUGs farther 3' (due to the higher frequency of Kozak sequences toward the 5' end and also scanning across other AUGs after initiating translation upstream; an exception to this is translation reinitiation where a ribsome starts at a second AUG after termination). In many instances, usage of downstream start codons is correlated to copy number of mRNAs, suggesting it's a probability game.

Alex Kochetov has done some nice work on this in recent years.

All this to say: In future projects I would think it is probably better to get rid of the GFP start codon so it will only be expressed as a fusion and you won't have to worry about alternative translation initiation mechanisms. Just be as careful as you can with the mouse you've already made. Maybe try localizing the fusion protein by IHC, though I'm guessing that's challenging and is why you made the GFP fusion in the first place.

Best of luck. Shoot me an email at [email protected] if I can be of any help.

Jonathan

I wouldn't worry about it, if it doesn't have a regulatory regions upstream. Otherwise any methionine would be a potential translational problem.

I would say, if possible remove the start codon for GFP. If you are making a trasngenic animal/mouse then you should definitely remove it. In case for cell transfections i think you can risk it, most likely you will not have any problem. So just give it a try without removal of start ATG of GFP.

My experience matches the comments here that internal translation starts for GFP fusions don't generally happen. However, proteolytic cleavage of GFP tags can occur on occassion. So if you observe a GFP-sized band with an anti-GFP antibody, this may be a cleavage product and not due to translation initiation at the start codon for GFP.

You should perform the suggested western blot above to detect if GFP is produced by itself. We had previously cloned GFP fused to our protein of interest leaving the start codon intact and ended up with high levels GFP in addition to the fusion protein. We had to go back and mutate the start codon of GFP in order to produce only the fusion protein.

It is a good idea to test expression of your fusion protein in cell culture prior to embarking on a mouse project just to be sure of expression levels.

I agree with Eric - check by western blot and see what you're getting expressed, checking the proteins cellular location by IHC/ICC using both the anti-GFP antibody and a protein specific antibody would also be useful (if you know its normal location in the cell). As you say, the mice are already made and they're an expensive investment so it's more important right now to know what your looking at in your current model. If you ever make a similar model in the future, just to be on the safe side, I would always remove the GFP start codon (or the start codon of any C-terminal tag in a fusion protein) for either transgenic model production or cell culture experiments.

After more than 20 constructs with the the start codon in both the endogenous gene and GFP, I would say it does not matter whatsoever.

It is not a problem as long as both, your gene and GFP, are in frame. The start codon will just be another methionine in the protein. What I would do first is to confirm that the protein product of your gene is functional and GFP does not affect it. You can test depending of the functions, a kinase, phosphatase reactions, a membrane protein by cell transfection, etc.

Hi,

I used a similar construct many times with both the start codons intact. Nothing really creates a problem. As already mentioned, if you are expecting a fusion, most likely the start codon of your protein plus if there is a kozak, would be efficiently used.

If you just make sure that a fusion is being generated by either sequencing your gene plus GPF fusion, you could easily make sure of the same and for expression western always is a good check.

It should not be a problem theoretically as eukaryotes should start translating from the first start codon. However in my experiece with similar clones I have observed some amounts of GFP without my sequence which I atribute to faulty initiation of translation or unstable mRNA in some clones. So to be absolutely sure do a western blot or some microscopy studies.

Since Met codons are present throughout genes, I don't see that it should be a problem. I have used several GFP fusions (one a fusion near the N-terminus) with both start codons present, and have not yet had any problem, judging by expression levels, protein size and protein function. All of these parameters- expression level, expression of intact protein, and function of protein fusion- should be checked anyway, no matter whether you are worried about a second start codon or not.

Agree with Daniel Cohen.

I have met a similar situation. Fuse AzamiGreen (AG) to the gene of interest at the C-terminus without removing of the start codon of AG and introduce it into human cell lines. It worked. Good Luck !

It depends on the assay, but in most cases, the presence of untagged protein due to any infrequent, spurious translation initiation events will not interfere with the assay.

Thanks for all the responses! Once we have a stable line we will test the transgenics with a western targeting our protein of interest and also one targeting GFP. Thanks for relieving my worries in the meantime! :)

-val

Given what you said in your introduction, the EGFP start codon is here, in effect, a Met codon. For a C-term tag, the start codon of your tag shouldn't be a problem, and as you stated that you removed the stop codon from your protein of interest, I'd say you're good to go. To pile on on what Toufic wrote above, it is worth noting that free GFP might arise following partial proteolysis of your tagged protein. If, for example, your protein is, under some conditions, a proteasomal substrate, it might be degraded up to the GFP moiety. This moiety has a very tight fold which makes it hard for the AAA ATPases of the 26S proteasome to unfold. As a result, you might observe some free (or, rather, freed) GFP on a Western-Blot.

Hi Valerie

About your question is very good to ask it Because I have met a similar situation before in my experiment .

I agree with David Chiluiza`s answer that your gene and GFP, are in a frame.

My experiment was rescue experiment and GFP expression is very important to me because I found my gene already has expressed in the model system but GFP not and I changed to RFP and BFP and the problem was still with me so I sent my plasmid construction to campany to give me sequencing and my seq results is right so I changed my Splicing Factors that I used it with GFP (at first time I used Sl-1d) to Sl-2d :: GFP and my GFP already has expressed and finished my experiment.

Splicing Factors that I used it with GFP (at first time I used Sl-1d) to Sl-2d :: GFP and my GFP already has expressed and finished my experiment.

what does it mean?

and how it can be related to GFP expression. I am going to make such construct so i am asking.

Thanks