Hi Monika,

Is this a promoter that you're trying for the first time? It’s hard to predict whether the region upstream of a certain gene includes all the regulatory sequences required for a reporter line to reflect the gene’s endogenous expression pattern. Did you try amplifying fragments of different sizes? Or perhaps including the first exon and intron in your entry vector? You can check if the construct is working with an RTPCR (just make sure your RNA sample is free of DNA contamination before RT). 

If indeed the promoter is working, but you still see no signal perhaps you could try using a very strong Kozak sequence, a 3’UTR and polyA that gives you higher stability, using a brighter fluorescent protein or even several fused copies of the fluorescent protein to increase the signal. Also, you could switch to using a driver system. Did you also check for fluorescence in a confocal microscope?

If you see that the integration is good (I assume because you have a marker gene in your construct), but you still see absolutely no signal where you expect it, I would rather think it is a problem of the promoter itself, and waiting for the F1 might not improve anything. You could try incrossing the F1 to have more copies of the transgene in the genome.

Hope that was helpful… good luck!

Hi Monika,

Is this a promoter that you're trying for the first time? It’s hard to predict whether the region upstream of a certain gene includes all the regulatory sequences required for a reporter line to reflect the gene’s endogenous expression pattern. Did you try amplifying fragments of different sizes? Or perhaps including the first exon and intron in your entry vector? You can check if the construct is working with an RTPCR (just make sure your RNA sample is free of DNA contamination before RT). 

If indeed the promoter is working, but you still see no signal perhaps you could try using a very strong Kozak sequence, a 3’UTR and polyA that gives you higher stability, using a brighter fluorescent protein or even several fused copies of the fluorescent protein to increase the signal. Also, you could switch to using a driver system. Did you also check for fluorescence in a confocal microscope?

If you see that the integration is good (I assume because you have a marker gene in your construct), but you still see absolutely no signal where you expect it, I would rather think it is a problem of the promoter itself, and waiting for the F1 might not improve anything. You could try incrossing the F1 to have more copies of the transgene in the genome.

Hope that was helpful… good luck!

 That's fantastic, thank you!

Hi Monika,

          The suggestions from Paola were all great. Did you consider some trivial things that we sometimes overlook. Are you checking for expression of your promoter at right time in the right tissue. Did you use correct filters. While there is evidence that germline integration is independent of somatic integration, it is important to feel confident that your promoter is working correctly for you to wait that long. If you cannot get the promoter to work an alternate strategy would be to BAC transgenesis. It is rather tedious but BACs tend to contain most regulatory sequences (of course there is no certainity that they will have all the regulatory information). If you need info on BAC transgenics I can send the protocols I use. You can also try to knock-in the fluo protein into your gene of interest. I haven't tried it yet.

Good luck

Sandeep

PS-Another important criteria is the quality of transposase RNA. In our hands it is the single most important factor determining the success of getting stable lines...

Thank you for your answers! I am not creating stable lines, I am just checking the activity of human promoter in zebrafish development. I have a positive control, using the same transposase, so I suppose the quality is good enough for it to cut the plasmid. 

One of the issues I'm dealing with, however, is imaging. I might not have seen the activity as I might have missed it or not detected it with the microscope.  As I don't know where to look, I need to screen the whole fish and I do not know how to do that, without bleaching the signal and/or affecting its development?

Are you using GFP for fluorescence readout. The current eGFP is extremely stable and very hard to bleach with a regular stereoscope. Even with confocal you would need a fairly high laser power to bleach it. Also eGFP is stable for several days; we even used it as a short term lineage tracer after the promoter shut off. I feel that your FP is not being expressed. I would recheck al cloning stages, sequence information etc...Also if you have a transient transgenesis marker (a-Cryaa:CFP) it should tell that your plasmid is integrating. I prefer not to use cmlc2:GFP as a transgenesis marker because the cmlc2 promoter is extremely strong and might repress other promoters in the vicinity.

I am using mCherry as a reporter. The sequence that should drive it is a human promoter, characterized recently in cell culture. So, to be honest, it might not work in zebrafish ;) But I do have a "feeling" that I should try better with proper imaging technique.

I have checked the sequences, made sure the promoter doesn't mess up mCherry start site, I have no out-of-frame occurrences.

Could you explain a bit more about the transient transgenesis marker, please?

Thank you very much!

It's hard to be sure but many human promoters/enhancers work really well in fish. We've had a collaborator make hundreds of lines with human enhancers in zebrafish. It is very easy to see ectopic expression in transient injections (especially in muscle) hence I am surprised that you don't see anything.

Since you mentioned tol2 I assumed you were using tol2 kit from Chi Bin Chien (http://tol2kit.genetics.utah.edu/index.php/List_of_entry_and_destination_vectors). Also check this paper

"The Tol2kit: A multisite gateway-based construction kit for Tol2 transposon transgenesis constructs"

The kit uses uses three-insert multisite Gateway cloning (att4-att1-att2-att3) from Invitrogen. There is just too much information for me to write in but you should read up on the link I shared above. In brief, you clone the promoter, gene (mCherry) and polyA in 3 different vectors (called entry clones) using BP cloning. Once you have the clones' sequence verified, these three elements can be added in correct sequence to a destination clone that contains tol2 arms for transporting the "cargo" to genomic DNA. Within the tol2 arms but outside the entry clones these destination clones may have cmlc2:GFP or a-Cryaa:CFP "transient" transgenesis gene. This maker will give you heart:GFP or lens:CFP expression telling you that your cargo has inserted in the genome. This is especially useful if you are making GAL4-UAS or Cre lines.

The Tol2 kit contains many useful constructs already assembled and you can just get it as a complete set. Most fish labs have it.

I am using a p5E-mcs-PCR and pME-Cherry , which I simultaneously cloned in LR reaction into pminTol-R4-R2. It's a three-way Gateway recombination, but you only need high quality LR enzyme, instead of BP and LR. I've cloned my promoter region into p5E, which means it is upstream of mCherry and both of them are inserted onto minimal Tol2 backbone.

For the positive control I have done exactly the same, except I've used pENTR5'_ubi instead of p5E. This one worked beautifly and I have gotten lovely mCherry mosaic expression in muscles. Unfortunately with my promoter sequence I've not seen anything.

I was thinking it is either too specific for me to detect it, it works only at a certain developmental stage or it doesn't work at all.