Dear Domenico,

You could try to express an epitope-tagged version (Myc, His, HA, V5,...). There are pCS2-vectors for this available via Addgene. Have you tried overexpression in a cell line as a control. It could be that the protein is stopped by the cell's quality control and leads to UPR and elimination of your protein.

The fact that the CFP-fusion does not work is not a good sign, but maybe expression of a less bulky tag will help.

Kind regards,

Jo

It depends what you are trying to do. We typically put the protein or protein fusion with GFP or a tag into the vector. If you get overexpression you will be able to trace it fluorescently or through antibody to the protein/fusion tag.

Hi Domenico I think all of the previous comments were good. Do you know for sure your flourescent fusion is functional? Where you albe to detect your mRNA after injection via RT-PCR or in-situ hybridization? This might give you a clue as to where and when it is being expressed and areas to target for looking for your expressed protein. Do you know anyting in terms of how stable the protein is? Can you resuce or alter your phenotype by injecting mutant mRNA variants?    

I have similar problem of detection, but using tol2 system. I would try to clone and transcribe just a fluorescent protein first as a ctrl. What stage do you micro-inject embryos and when do you extract proteins? From my experience, micro-injecting of mRNA into the embryos gives you very short-term expression and also do not forget that your mRNA is diluted to many cells after organism development. My guess you will not see anything in 4-5 days after injection even if you inject a X ug/ul of mRNA.

Thank you very much for your answers. I'll try to answer all questions.

First:injection experiments. All embryos were injected at one cell stage and screened for fluorescence at 24 hpf; the same stage was used to extract protein to perform western blot analysis and immunoistochemistry.

Second: Do I have good antibody? The answer is yes, my lab has good antibody both for western blots and immunoistochemistry, that works well in cells. But when I use these antibodies to detect overexpression both through western blot or immuno, I didn't see any diffrerence (always at 24 hpf).

The RNA on a gel is fine, I did not see a huge smear, and the band is of the right weight.

Concerning the fusion protein translation, I sequenced the plasmid and everything is fine. I made the tol2 final vector by generating a middle entry vector CFP-gene just by cloning from a pcdna3 containing the CFP fused to the gene. If I inject pcdna3 CFP-gene of interest, I can see fluorescence, but positive cells are too low, expressoin at several concentrations is too low to make experiments, that's why I tryed another approach.

I also tryed to rescue morpholino injected embryos, but I couldn't rescue the phenotype. 

This morning I have transfected cells with my cfp-gene construct to see if it works in HeLa cells. 

I hope you can help me figure out what's going wrong, because I have two years work to save....

I do not understand about tol2: you injected the same transgene in tol2 and did not see the expression? For tol2 you always have the mosaicism, you need to develop a transgenic line with good expression first.

For cells you can transfect them with a plasmid (if you have cmv promotor), no mRNA need. BTW, do you have some papers on mRNA injection to the embryos? I have injected only RNA transposase or gRNA  and mRNA for CRISPR/Cas9 system, both work at early stage. Cas9 fused with GFP - did not detect any fluorescence at all. 99% sure that mRNA is OK (checked on gel).

To check if my gene was expressed, I tryed several approach:

-western blot and immunoistochemisty (didn't see any difference)

-injection of cfp fused to my gene

I have injected my gene fused with cfp from 3 different plasmids:

-pcdna3: I can see CFP, but very few cells per embryos

-pcs2+: if I inject the plasmid I see so much expressoin as pcdna3; if I transcribe RNA I don't detect any fluorescence (RNA is fine on a gel)

-ptol2 vector: I could not detect fluorescence.

So my question is: is a problem of protein expression? Since cmw expression is low, maybe the tol2 system, restricting gene expression to only some cells, reduces the expression of my protein?

Dealing with papaer on RNA, I have papers, but I can't understand what's going wrong.

Thank you Jesus for your idea.

So dealing with pcs2+ and pcdna3, the promoter is a CMV, so an ubiquitous one, but the level of expression of my transgene is very very low (I mean number of positive cells).

The Tol2 vector is composed by an UAS promoter which control expression of CFP-Gene of initerest. I have injceted this plasmid into a trangenic line which express Gal4 ubiquitously under the control of an heath-shock promoter (hsp70), without seeing any fluorescence after 4 heatshock at 37 °C (from 24hpf to 48hpf). Then I injected the same tol2 vecotr in the transgenic line I use for my experiments, whcih express Gal4 in Rohon-Beard neurons: although I can detect a UAS:GFP correctly, no CFP-gene of interest is detected. 

The interest thing is that today I imaged the cells I transfected with a cmv gal4 and the tol2 vector expressing UAS-CFP-gene and IT WORKS in vitro. So tomorow I'll try to injected different concentrantions in different lines to see if it was just a problem of concentrantions level.

You said "I have been injecting mrna (WHAT SORT of mRNA and WHERE DO YOU INJECT) for two years, making experiments with this mrna and finding interesting data WHAT DO YOU MEAN BY INTERESTING RESULTS)". When you ask advices you have to be more accurate.

Did you verify that after injection that you mRNA( or total RNA or PolyA+ RNA) was not rapidly degraded???

mRNA was transcribed from a TOPO blunt vector, where my gene followed by a bgh polyA was cloned. mRNA was injected into one cell stage eggs in the yolk. By interesting results I mean that that mRNA produced an effect on the transport of mitochondria. I did not verified RNA degradation. Do you mean by RT-PCR or something similar?