Rescue transgenic display different expression pattern

Hi All,

I have transgenic C. elegans strain transcriptional reporter for gene X ( gene X promoter::GFP) showing expression of gene X in specific cell bodies of neurons.

I generated transgenic rescue C. elegans strain for gene X under its own promoter (gene X promoter;gene X cDNA::GFP)
which display expression of gene X in pharyngel muscle and not in cell bodies of neuron.

So, I am not finding expression pattern similar in my Transcriptional reporter and translational reporter (rescue) transgenic strains.

My questions are

  1. Why the expression is different?
  2. Did anybody faced similar situation?
  3. How to trouble shoot this ?

Will appreciate your replies.

Thanks for your time and help
S. Durai

Transcriptional reporters can be used to rapidly establish a tentative expression pattern for a gene of interest. Fusing 5′ upstream sequences to GFP can be done in a number of ways and usually presents no technical challenge. Compared to translational and smg-1-based reporters, however, promoter fusions may not give a complete representation of the real expression pattern of a gene, both spatially and temporally.

Translational reporters can provide a faithful representation of a gene’s expression pattern because additional regulatory information that may be present in introns or 3′UTRs is included in such reporter constructs. When mutants are available, translational reporters can be used in rescue experiments. Successfull rescue of the mutant phenotype lends support for the functional relevance of the resulting expression patterns. In addition, translational gene fusions can also provide information about subcellular localization and the temporal aspects of gene regulation. Translational fusions may appear less bright than transcriptional fusions due to the intrinsic instability of the protein fused to GFP. However, insertion of GFP intragenically can sometimes disrupt protein function or even lead to toxicity of the chimeric product. Finally, translational reporters that exhibit subcellular localization can make cell type identification more difficult because the shape of the cell may not be visible (especially for neurons).

Yup, what Snug quoted. Two other additional points/thoughts:
-if you’re using cDNA of gene X, then you might be missing regulatory elements in the intron(s). Also, are you using the 3’UTR of gene X or another one? Lack of introns or the appropriate 3’UTR could also lead to a failure to recapitulate the true expression pattern of gene X
-if your question is where is gene expressed/protein X localized, I’ll give what seems to be my most frequent suggestion: why not just CRISPR it? Use the Dickinson/Goldstein SEC vectors and N-terminally tag gene X. You’ll create a promoter reporter first, and then SEC excision will create a translational fusion, all in the endogenous locus. If for a promoter reporter, you were unable to identify cell type, you could do a C-terminal fusion and add an P2A and NLS sequence to the fluorescent reporter. Then you would get an NLS tagged fluorescent protein produced from the endogenous mRNA, which should give you an excellent readout for what cells in which the mRNA is expressed.

And another echo of Jordan’s comments. We have already found two expression patterns by CRISPR tagging that was not there by promoter::GFP fusion. In both, the promoter::GFP did not express in the tissue where we were confident the protein functioned. The CRISPR tag of the endogenous gene, however, was expressed where the phenotype and rescue (in one case) said it should be.

one other thing to keep in mind: I was working on a gene that I was pretty sure acted cell-autonomously (because of what the gene encoded, and because a cell-autonomy experiment looking at array loss said if it wasn’t acting in the cell, it was acting in a cell incredibly close by lineage), but a rescuing gfp transgene, made by dropping the gfp open reading frame into the genomic rescuing construct at various places until one rescued, didn’t give fluorescence in my cells. Until, based on someone’s advice (and sadly I can’t remember whose), I looked at the cell in the embryo, when the gene had to function, instead of in the larva, when the cell was easier to find.

Shorter version: it helps a lot if you know the gfp reporter rescues (either as a fusion or as a bicistronic construct; you say you made a fusion, but don’t say it actually worked), and if you still can’t see the expression you might want to look at a different time point, or look with more sensitivity, for example with an anti-GFP antibody.

Also, specifically for your pharygeal muscle expression: try removing the plasmid backbone prior to injection; the plasmid backbone of the Fire vectors can drive expression in the pharynx.


Thanking you all for your valuable suggestions, I am working on this taking in all your comments.