Hello -
My first post in a forum I’ve been using as a valuable resource for many years! Thanks for all the great insight. Hope you can help with this one.
I’ve recently made a transcriptional reporter with eGFP. It seems to localize primarily to neurons, and perhaps some hyp or muscle. The GFP seems strongly
excluded from the nucleus, but also seems localized in globs within the cell bodies. It’s not punctate, more reminiscent of mitochondria (although I am not super
familiar with neuronal mitochondria, and if they would look the the classic body-wall muscle mitochondria). But that would make sense given the gene.
But there is no protein! Why would a transcriptional reporter behave this way?
Nuclear exclusion (perhaps fast export?) and not smooth, even localization throughout the cytosol… Are these just artifacts of neuronal expression? It looks
similar in 4/4 lines.
Are you sure it’s just a ‘transcriptional’ reporter? Sometimes, people fuse the first few eons to GFP to manage some large introns that might have promoter elements. Did you do this? There may be a mitochondrial signal peptide in the coding sequence, or from an unannotated start site, that is being added to GFP, unless you made the reporter in some other way.
Assuming you’ve been careful with respect to the annotated gene structure, I like Kevinem’s idea that there’s a non-annotated start site providing additional sequence that contains a localization signal.
Maybe try 5’ RACE with gfp sequence? Or introduce a frameshift upstream of the start codon you think you’re using?
I too like kevinem’s idea and Hillel’s suggestion. But before doing the 5’ RACE yourself, you can check the Fire, Meyers and/or Ahringer Transcriptional Start Site datasets to determine if there is any alternative start sites that have already been annotated. I have .bed files for those datasets that you can visualize in a genome browser such as IGV, so PM if you would like those files.
Thank you for the comments! Some good food for thought. It should be a straight transcriptional reporter,
constructed via 3-way Gateway. I will look more into the promoter I used…
Interestingly, I recently found a very similar pattern when I made a transcriptional reporter fused to GFP. When I used MitoTracker to check for colocalisation, it showed that it was not being targeted to the mitochondria. The expression is present in the muscle, seam cells, intestine and nerve ring. Again, in 4/4 lines, I see the same network like appearance in the expression pattern. When I looked into the gene sequence further, I found a putative exon which may be targeting it somewhere. It might be that there is are additional exons in the promoter which are causing the unusual pattern!
Interesting! Can I ask how you identified the putative exon? This is just not something I’m familiar with! Do you have any idea what organelle (?) it might be localizing to? Thanks in advance!
To identify the putative exon, I looked at the gene sequence of the promoter on snapgene and found that there was a 400bp sequence which could be translated in frame with the gene I’m studying. I have recently repeated the MitoTracker colocalisation experiment using a different protocol and I have found colocalisation! I grew the worms in a liquid culture of HB101 bacteria in a 24-well plate containing 2ug/mol MitoTracker Red CMXRos (In-vitrogen) dye and incubated them for 24 hours (shaking at 70rmp at 20 degrees.) I then washed them and allowed them to crawl for an hour on an NGM plate then washed again with M9 and imaged them on a confocal microscope. I hope that helps!