Earlier this week we had the chance to tour the Zon Lab at Harvard Medical School / Boston Children’s Hospital. Katie Kathrein, our instructor from Cell Biology this spring, is a postdoc there and very generously offered to show us around and chat with us about what zebrafish could do for our cause. They have an amazing setup and I got some awesome pictures:
(Or just follow this PicasaWeb link if the Flash doesn’t work in your browser)
Zebrafish, or Danio rerio, have become a popular model organism, particularly for studying cancer and developmental abnormalities. This is thanks in large part to Dr. Zon himself, who I’ve heard people refer to as “the zebrafish guy.” Zebrafish are fairly transparent, so you can actually see blood pumping through their hearts. In their youth, they’re small enough to fit ~10 of them into each well of a 96-well plate, meaning you can do high-throughput screening on whole organisms. Transgenes integrate easily into their genomes without viral vectors – the DNA just integrates into a chromosome on its own.
Conversely, it’s relatively difficult to do knockin and knockout because their homology-directed repair mechanisms are either not very active or just not very well-understood by us. Instead of knockouts, people use morpholino to knock down genes. Somehow, you can inject antisense morpholinos into a zebrafish zygote at the single cell stage and this will result in stable repression of a gene’s expression even after the cell divides and grows into an embryo – I’m still not clear on how that actually works (are they just putting in a ton of copies of the morpholino??).
Zebrafish have two genes that are considered to be orthologs of mammalian prion protein, dubbed PrP-1 and PrP-2. These genes are quite different from mammalian PrP but there are still a few stretches of very tight homology (including the area around human codon 178). From what I’ve seen, most (perhaps all) research being done on zebrafish PrP right now is oriented towards exploring PrP native function [reviewed in Malaga-Trillo & Sempou 2009, Syed 2011]. Ted Allison from U. Alberta also had some posters up on his lab’s native function research at Prion2013.
Personally, be quite curious to really know PrP’s native function (“well this better be good”) but it’s not a high priority for me. Whatever the native function is, it appears to be dispensible - or at least, losing it couldn’t possibly be worse than prion disease. Instead, my interest is in figuring out ways to deplete PrP.
We and Katie discussed the ways that zebrafish could be a useful platform for working on this goal. One possibility was whether we could use a whole-organism zebrafish screen in 96-well plates to look for compounds that reduce PrP expression. For instance, if you wanted to screen specifically for compounds to inhibit PrP transcription, you could introduce a visible transgene (GFP, luciferase etc.) under the control of the human prion promoter and then screen for compounds that reduce the fluorescent/luminescent signal. Whether this would work well (or at all) would hinge on whether the transcriptional regulation of PrP is conserved between zebrafish and humans. The first step on my to do list is to check how homologous the zebrafish prion promoter(s) are to the mammalian prion promoter. If the promoter sequences are highly conserved, it would seem likely that they’re under the same regulatory influences.
Paradoxically, though, any mechanism (transcription factor, etc.) that is so highly conserved that we share it with zebrafish seems likely to also be so biologically important that it’s not a viable drug target.
Incidentally, mammalian PrP can rescue PrP loss-of-function phenotypes in zebrafish [Malaga-Trillo & Silos 2009] but this work was done by directly introducing mRNA; I’m not aware of any use of mammalian PrP transgenes complete with the mammalian prion promoter in zebrafish.
A while back we got a broad outline of the timeline required to run a chemical screen in induced pluripotent stem cells - figure about a year total, though as in any screen, it’s all prep time and the actual screen takes under a week. Same with zebrafish, at least if you’re using a transgene for your readout. Katie said that just the effort of getting a transgene in and then breeding them up and crossing them to make sure you got stable transgene integration and copy number takes several months.