How do you mutate zebrafish so that they can be used as models of human diseases, like Duchenne muscular dystrophy?
After watching this video, following up with Part 2.
Don Love (Auckland University): So I wish to modify gene expression in a targeted way within the fish. Could I perturb a particular gene which would play a critical role in disease development?
There are many ways you could do it, one of which could be random chemical mutagenesis, where we treat fish with a chemical mutagen to give rise to mutant sperm, let’s say. And those fish are then mated with normal females. You have no control over the mutation event. There is going to be a whole slew of mutation outcomes and you would need to identify among hundreds of thousands of mutants the one you are particularly interested in.
Peter Cattin (Auckland University): We’ve conducted a few new chemical mutagenic screens here, where we’ve exposed the males to a very low dosage of a chemical mutagen for long enough for that mutagen to get into the germ line. We then spawn those males. The first spawn we discard because it won’t be fixed in the germ line at that time. Subsequently, we collect what is known as mosaics [which contain a range of different mutations]. We then strip the females; we outbreed them through a stripping process which is where the eggs are stripped from the adult female, and the sperm is stripped from the adult male - which you can appreciate from small fish like this is quite tedious and needs to be done very carefully. Then we do an in vitro fertilisation. Then from that we look at the phenotype of the developing embryos under the microscope, looking for carriers of the particular mutant that we are interested in. And then we will grow that out.
If it doesn’t survive, which is often the case, then we look to the combination of adult male/ female that produced the phenotype of interest and try to reproduce it. It is a fairly tedious process and the success rate is relatively small. You only get about a 3% mutant that you are interested in.
Don Love (Auckland University): The second one would be, can we target a particular gene? Can we knock out a gene - only one, not the other thirty thousand, that one in particular? And that is what we are working on now.
In terms of our work so far on replicating some aspects of Duchenne muscular dystrophy, we have targeted the critical gene encoding the protein responsible for this disease, and we do see some outcomes affecting muscle. We see changes in muscle function, muscle structure, [RNA] transcript changes, and we can reveal a certain aspect of the human disease that has been overlooked thus far. And so it can give you a window of looking at events that you haven’t seen in humans, but would be there if you looked hard enough.
I see pathophysiology as the end point of a long series of events, and all that has to be replicated with my model species. What if they’re not? What if the early events are replicated in my species compared to humans, but after that things get a bit fuzzy? I’m sorry, the pathways are different. It’s a fish, after all. In which case I might not see the same outcome. So what outcomes do I measure? Well, as a molecular geneticist I love transcripts - RNA - so I measure transcript outcomes. If I target a particular transcript, can I measure that level [in my model]? [If] the transcripts are okay … I can see similarities … proteins – yes … I can see a similar sort of response … patho-physiology, I might not.