Hi everyone. Currently, I cloned a construct of ISce-I_ubi promoter-Gene X_2A-mCherry_ISce-I (the backbone vector is pDestTol2CG3 with cmlc2:GFP as a 2nd reporter). I injected the construct in zebrafish embryos and found the expression of mCherry in muscle cells, different levels on different larvae. Since they are mosaic at this stage so I am not worried. I also did WISH against the transcript of gene X and found ectopic expression in cmlc2:GFP+ larvae so it seems to again validate the integration event. Also, I did antibody staining and see some cells also expressed the protein in the same positions with mCherry.
I raised my injected embryos to adults. Outcross them with AB and screen the embryos. So far, I only got embryos/larvae with cmlc2:GFP+ and not mCherry expression at all! This is a big surprise for me. I tried to bring the cmlc2:GFP+ to confocal but also don't see mCherry expression in other cell types. Also, antibody against gene X is not detected anything.
Could you share with me some experience here? What can be wrong and how can I fix it? There are some more fish to screen but I tried about 5 injected fish already. is the construct bad? In case I have to do again, could you suggest other promoters expressed ubiquitously?
many thanks,
Hung
Did you remove the ATG from the mCherry sequence after the 2A peptide? This can reduce the rate of false positives in the F0.
Hi,
I had the exact same problem using the ubiquitin promoter to drive my BFP-tagged transgene and cmlc2:GFP as a transgenic reporter. I got a few embryos that were expressing BFP in the F1 (out of many that were expressing green heart), but after raising them and outcrossing all the progeny were BFP -ve. Even after repeating the same cross that gave me BFP +ve embryos previously I wasn't able to get BFP embryos again. I confirmed the integration event by PCR, however was getting no expression of BFP or my transgene. We are not alone with this experience.
I think there are multiple reasons this could be happening. The transgene could be getting inserted into transcriptionally silent genomic loci. As cmlc2 is a strong promoter it could be resisting silencing, whereas ubi does not. This is unlikely if you have screened plenty of founders as at least in human cells Tol2 preferentially integrates into open chromatin (Grabundzija et al., 2010).
You could also be getting insertion of naked DNA, which tends to form concatamers and mess up your construct as well as making it more prone to silencing (Kawakami, 2005). This is also unlikely if your Tol2 mRNA is good quality and you are getting a relatively high integration rate.
Another speculation of mine is species-specific methylation, but I haven't found much information on this and it shouldn't be a problem if your gene is a zebrafish gene.
Finally, there is some anecdotal evidence (reported either by Kawakami or Chen labs, can't remember now) that cmlc2 promoter may actually repress ubi promoter itself.
My suggestion would be to either change the promoter, change cmlc2 marker (or remove it altogether) or start using the zebrafish gene if you aren't already. Sorry there doesn't seem to be a quicker way.
Hope this helps. Unfortunately, I didn't have time to apply the suggestions myself anymore, but if you do try any of them, it would be interesting to know what results you get.
thanks Filippo Del bene and Ringaile Zaksauskaite
In my previous constructs, the ATG is still in mCherry.
I have other constructs with gene-X driven by l-fabp10a and tp1- promoters and with different fluorophores (GFP and H2B-mCherry). I still used the same cmlc2 as 2nd marker. I'll see the results in next few weeks.
- Badges
- Science topic