If you haven't already, the first thing you should do is confirm successful knock out of your gene/s. If a specific antibody is available, confirm the absence of protein by western blot or, even better, confirm its absence in skeletal muscle using IHC. Can you detect evidence of nonsense-mediated decay by in situ hybridisation?

If the knock out is successful, then you can explore potential reasons for the lack of phenotype, where one might be expected. One possibility is compensation by paralogous genes with similar function. Can you detect an increase in mRNA transcripts which might be compensating? Are they expressed in muscle?

Another possibility is that you are missing a phenotype. How are you looking for evidence of poor sarcomere assembly? You should, for example, stain for a range of myofibrillar proteins (e.g. MyHC, actin, alpha-actinin, titin.... etc)...

Cheers,

Mike.

Michael Attwaters Hi Mike, Thanks for your nice reply!

Now I got two mutant lines of this gene. One line has nonsense RNA decay, which is knocked out in the middle exon of the whole gene. And another line does not have RNA decay, which is knocked out in the 7th exon/9 exons in total. By the way, can the mutant line without the nonsense RNA decay be viewed as a successful knockout?

As for western blot and IHC, there is no specific antibody used in zebrafish before based on previous studies. I have not tried yet.

I have done some antibody staining for myosin, actin, a-actinin, there was no phenotype yet. Maybe next I will do RNA-seq to find some potential reasons .

Hi Huanhuan,

I hope you are staying safe during the Covid-19 times.

You might predict that your exon 7 mutation would escape nonsense-mediated decay (NMD), since the translating ribosome will evict all exon-junction complexes (EJCs) as it traverses until exon 7. (Recall that EJCs are thought to be signals to the NMD machinery to degrade the transcript.) What was your rationale behind targeting Exon 7? Targeting late exons can lead to C-terminally truncated proteins that may be functional. Your middle exon mutant might be better in this regard and it is particularly encouraging that you see evidence of NMD.

You could try some human antibodies and see if they cross-react. If you can, find out the epitope sequence of each available antibody and align with the zebrafish protein; >90% homology might work.

Michael Attwaters Hi Mike, hope you stay well too! Thanks for your kind reply!

Yes, I also found that NMD did not happen when I targeting the exon 7 recently, leading to a truncated protein. When I knocking out this gene, I did not realize this issue, I just designed several sgRNAs in different exons(functional area). Now I found the mutant line generated by sgRNA in the middle exon, there are some phenotypes. But the line generated by sgRNA in the 7th exon did not show the same phenotype. Then I realized this issue. I think maybe the truncated protein still has some functions. I am going to injecte a trucated construct to see if this phenotype cused by the middle sgRNA can be resued.

The antibodies used in mouse about 80% homology with zebrafish. I can have a try.

Thanks!😊