Model Organisms in Drug Discovery

OUTLOOK: THE FUTURE HAS STRIPES 197
most cases a combination of approaches is taken. The candidate gene is
sequenced in both its wild type and mutant allelic form. Moreover, if the
injection of a phospho-morpholino against the candidate gene can phenocopy
the mutant phenotype, then this is a strong indication that the correct gene has
been found. Also, expression of the mRNA of the respective gene should be
detectable at or before the stage where the phenotype becomes apparent and
ideally is restricted to the tissue affected by the phenotype. This final step can
take anywhere from 2 weeks (in those cases where multiple mutant alleles are
available and all of them carry convincing molecular lesions) to 2 months (in
those cases where a phospho-morpholino needs to be ordered and the
mutations are difficult to identify on the molecular level).
Although none of the technologies necessary for the positional cloning
approach outlined above are unique to zebrafish, there are a couple of specifics
that should be borne in mind. Unlike in other vertebrate systems, it is
comparatively easy to collect a few thousand mutant embryos. Consequently, it
is possible to let the fish do much of the ‘genetic work’, such that fine mapping
with a very high degree of resolution allows a quick narrowing down of the
interval in question. The downside to this approach is that one needs to wait for
an entire generation time until one is in the position to start collecting
homozygous mutant mapping embryos. Therefore, with any positional cloning
project one will never be able to push the time-lines below the biological limits
of generation time. However, the molecular work will, in years to come, become
more efficient and will be supported by more complete resources such as
libraries, expanded marker sets and the zebrafish genome sequence. This will
considerably decrease the time-lines for positional cloning projects.
7.7 Outlook: the future has stripes
Zebrafish have evolved rapidly from a pet-shop inhabitant to a widely used
genetic and experimental system. The times are long past when zebrafish
researchers unvaryingly started their seminars by explaining why they work on
zebrafish. The available resources and technologies that have been developed
in zebrafish over the last few years are truly impressive. More development,
however, is still needed. For example, setting up large-scale genetic screens
where thousands of embryos or larvae are scored on a daily basis for 6 months
remains very difficult on the screeners. In this area any sort of automated
screening would be highly desirable. Semi-automated image capturing can be
envisaged for at least a number of assays and would be a step forward in terms
of time-lines and labor costs for a screen. Another area that would benefit
from shorter time-lines is positional cloning. Starting with a mapping panel
(48 or 96 mutant and sibling embryos each from a mapping cross) in hand,
positional cloning of a mutant can take anywhere from 3 months to 1 year.