Model Organisms in Drug Discovery

192 GENETICS AND GENOMICS IN THE ZEBRAFISH
chain reactions (PCRs), amplifying 2.7 kbp of a gene of interest, in this case
rag-1. Subsequent sequencing revealed 15 point mutations, one of which
resulted in a premature stop codon. Going back to the corresponding sperm
sample, Wienholds and colleagues established a stable rag-1 mutant line.
The method outlined above is the only one at present that allows a mutant
zebrafish line to be defined in a preselected gene. In contrast to the knock-out
technology in mice, it is impossible to predetermine which nucleotide will be
mutated, let alone the possibility of deleting whole exons. On the other hand,
the method will provide the investigator with a number of mutant alleles per
gene to analyze, which is often very useful. The method is scalable and,
depending on the number of sequencing lanes one is willing to run, there is no
a priori reason why particular genes should be untractable by this approach.
Importantly, the frozen sperm and the DNA constitute a resource that can be
used over and over again, making it necessary to generate this resource only
once.
7.4 Drug screening in zebrafish
There is yet another interesting twist to screens and phenotypes in zebrafish.
In recent years, an increasing number of laboratories have caught on to the
idea of testing the effects of pharmacological drugs on zebrafish embryos. In
hindsight, the idea makes perfect sense. There is a high degree of conservation
between vertebrate genes and, consequently, the physiological effect that a
particular drug causes in mammals should have a high chance of affecting the
orthologous target protein in zebrafish. This notion has been put to the test in
a number of cases and has been found to work in some instances. Interfering
with nitric oxide levels by nitroprusside or N (G)-nitro-L-arginine methyl ester
(L-NAME), for example, results in changes in vessel diameter when applied to
zebrafish larvae (Fritsche et al., 2000). A complete loss of all vessels was
reported by Chan et al. (2002), who used the tyrosine kinase inhibitor
PTK787/ZK222584 to block the activity of vascular endothelial growth factor
receptors. Warfarin, an inhibitor of hemostatic proteins in mammals, induces
bleeding in zebrafish (Jagadeeswaran and Sheenan, 1999), which is consistent
with the notion of warfarin inhibiting the process of thrombosis and
coagulation in both mammals and fish.
A particularly elegant example of the possible uses of drugs in zebrafish was
provided by Langheinrich et al. (2002), who studied the function of p53, a
protein known to cause cell cycle arrest and apoptosis in cells that are severely
stressed or have undergone DNA damage. Using morpholinos, they
demonstrated that the lack of p53, as such, has no detectable morphological
effect in zebrafish embryos, a scenario very comparable to mouse embryos
mutant in p53. However, when exposed to UV light (inducing DNA