Model Organisms in Drug Discovery

THE ZEBRAFISH TOOL KIT 191
the end of a positional cloning effort (see below) but also by paving the way
for systematic reverse genetics in these organisms. It was suddenly feasible to
study the function of a large number of vertebrate genes on the level of the
whole vertebrate organism.
From gene to mutant to function: targeted mutagenesis
One of the obvious shortcomings of zebrafish has always been the lack of a
specific technology that has made the mouse so useful: the knock-out
(removal) of genes via homologous recombination in embryonic stem (ES)
cells. It is of small comfort that zebrafish are in good company in this respect,
but it would be highly desirable to be able to eliminate genes at will and study
the resulting phenotype in a loss-of-function situation. The use of
morpholinos (see above) is helpful in those cases where an early-acting gene
is of interest, but the knock-down caused by morpholinos is transient (it lasts
up to 5 days) and does not generate stable mutant lines.
Establishing ES cells and keeping them in culture in order to be able to
attempt homologous recombination in vitro has been the bottleneck in
zebrafish and many other systems (there might be other bottlenecks down the
road, but for the time being this is the most eminent problem). Only very
recently was it reported that a primary spleen cell line from rainbow trout
(Ganassin and Bols, 1999) is able to support the growth of zebrafish
blastomeres in culture and to keep most of the blastomeres in an
undifferentiated state (Ma et al., 2001). Blastomeres were transplanted into
host zebrafish embryos and were able to populate the germline (Ma et al.,
2001). Thus, they fulfill one important requirement for ES cells. Further
experiments are underway to determine whether these blastomeres can
undergo homologous recombination in vitro (Paul Collodi, personal communication),
which would satisfy another important criterion. Interestingly, it
has been shown recently that injecting morpholinos directed against the ‘dead
end’ gene renders the injected embryos void of pregonial germ cells (Ciruna et
al., 2002). Such embryos would be ideal recipients for in vitro manipulated
zebrafish ES cells, because if the ES cells were to populate the germline, the
whole germline would consist of manipulated cells of the desired genotype,
thereby circumventing the nuisance of mosaic germlines.
In the absence of ES cell-mediated knock-out technologies, other means
were found to create stable mutant lines in genes of interest. Wienholds et al.
(2002) have reported a way of generating multiple ENU-induced alleles in a
gene of interest. They have mutagenized zebrafish males using standard
protocols (Pelegri, 2002) and generated a library of F1 males. Sperm samples
were taken and stored frozen, whereas DNA was prepared from the remainder
of the fish. Over 2700 DNA samples were used as templates for polymerase