Model Organisms in Drug Discovery

188 GENETICS AND GENOMICS IN THE ZEBRAFISH
The generation time of zebrafish is 3 months, which is short in vertebrate
terms. Adult fish are 1 inch in size and the housing costs are very low once the
initial tank system has been installed. The transparency of zebrafish until
stages where organogenesis is well underway or completed makes zebrafish the
vertebrate system of choice for forward genetic screens designed to investigate
this process. Phenotypes are easily identified and the underlying gene may be
subsequently cloned.
In addition to standard genetics there is quite an arsenal of genetic tricks
that can be applied to zebrafish, including the generation of haploid and
gynogenetic embryos (for review, see Kimmel, 1989), as well as novel methods
to carry out maternal effect screens (Pelegri and Schulte-Merker, 1999). Still,
the most common screening scenario still remains the induction of mutations
in the parental generation and breeding the mutagenized individuals until two
generations later. The F2 individuals are mated and the phenotypes can be
examined in a homozygous situation (see below). Mutagenesis is carried out
by utilizing gamma rays, retroviral insertions and, most commonly, the
chemical mutagen ethyl–nitrosourea (ENU). These methods will be compared
briefly below.
Irradiating post-meiotic sperm with x-rays or gamma-rays was the first
attempt to generate fish mutants in a systematic fashion (Chakrabarti et al.,
1983) and it was successful in terms of very efficiently generating mutations.
Mutation rates up to 2% have been reported (Chakrabarti et al., 1983).
However, many of the mutant lines have proved difficult to maintain and
characterize molecularly, because irradiation tends to induce large deletions
and chromosomal rearrangements. Other attempts to circumvent these
problems and to establish protocols that induce small deletions while
maintaining chromosomal stability have failed (Lekven et al., 2000) and,
unless one deliberately desires to induce deletions, other methods for
generating mutant lines are preferable.
Insertional mutagenesis has proved extremely useful in the case of P
elements in Drosophila. In this system, the mutagen consists of a transposable
element that inserts into chromosomal DNA and compromises the expression
or function of the gene and gene product. When successful, it is fairly
straightforward to identify the underlying gene, because the P element serves
as a tag that facilitates cloning. In zebrafish, the group led by Nancy Hopkins
has established a protocol that makes use of a pseudotyped virus that is
injected into blastula-stage embryos and inserts its genome into the genomic
DNA of the fish embryo (Amsterdam and Hopkins, 1999). In those cases
where the insertion happens to occur in a cell whose descendants become
future germ cells, the insertion is passed through the germline and will, in a
fraction of cases, mutate a gene to yield a detectable phenotype. The key
features of this technology are producing a high-titer viral stock and
genotyping the F1 fish in order to select fish with the highest number of