Model Organisms in Drug Discovery

THE ZEBRAFISH TOOL KIT 189
independent insertions. Any phenotype of interest can be characterized
molecularly with relative ease by testing which insertion tag co-segregates with
the mutant phenotype, followed by cloning the flanking regions of the
insertion.
Although it is reasonably straightforward with this approach to identify the
mutated gene once a phenotype has been identified, the low efficiency of
generating insertions has kept the zebrafish field from adopting this approach
broadly. In comparison with to the widely used chemical mutagen ENU (see
below) the frequency of generating mutations is only 5–10% (Pelegri, 2002),
which means that in order to obtain the same number of mutants as with an
ENU screen, one needs to maintain 10–20 times as many tanks and set up
10–20 times as many crosses. Because many investigators are not content with
identifying just one mutant and, ideally, would rather collect all genes essential
for the process under study, ENU mutagenesis has been favored.
The alkylating agent ENU has been used in many large- and small-scale
screens and an estimate of well over 10 000 mutants have been generated in the
three largest screens to date (Driever et al., 1996; Haffter et al., 1996; Odenthal
et al., Tu¨bingen 2000 Screen, unpublished). Adult male fish are bathed in a
solution of ENU, inducing mutations in premeiotic germ cells. These founder
males are crossed with females to generate F1 fish that are heterozygous for
the mutations induced in the previous generation. The F1 fish are crossed with
unrelated F1 fish that stem from independent mutagenesis events. Brother–
sister matings within the resulting F2 generation produce F3 egglays that are
homozygous with respect to the mutation induced in the parental founder
male. Naturally, there are many mutations per founder male and it is not
uncommon to uncover more than one mutant phenotype within a single F3
egglay. Despite the fact that it can be cumbersome to clone an ENU-induced
mutant, there are a number of reasons why ENU screens are popular: they
require very little expertise (compared with insertional mutagenesis) and ENU
is very efficient in generating single-locus mutations (compared with the low
mutagenesis rate using retroviral insertions and the large size deletions that
affect more than one gene). The high hit rate also opens up the opportunity to
identify, even with a middle-sized screen, a number of mutants that affect the
biological process under study and hence to identify a number of genes that
result in identical or similar phenotypes.
From gene to function: reverse genetics using morpholinos
With the ever-increasing number of publicly available expressed sequence tags
(ESTs) and the prospect of a fully sequenced and annotated genome, the lack
of reliable techniques to perform reverse genetics has become more evident in
the last few years. Approaches such as injecting antisense mRNAs made in