Model Organisms in Drug Discovery

196 GENETICS AND GENOMICS IN THE ZEBRAFISH
or MegaBACE (T. Wagner, personal communication) and a higher
throughput. Once a mapping pipeline has been set up (which involves
considerable work initially), one person can put two to three mutants on
the linkage map per week (P. Beeckmann and T. Wagner, personal
communication).
The information that one obtains from this initial mapping is very useful. In
cases where a lot of mutants are to be mapped, binning the mutants into
‘chromosomal groups’ tremendously reduces the amount of complementation
work that needs to be done to determine the number of genes, because only
mutants mapping to the same linkage group need be considered for
complementation crosses. Also, getting information about the rough position
of the mutated gene of interest opens the door for a possible candidate gene
approach, where candidate genes in the vicinity of the mutant locus can be
considered for further linkage analysis.
The second step in a positional cloning exercise consists of defining the
closest markers left and right of the locus of interest. To that end, all
available markers in the region determined in step one are tested for linkage
on a single embryo basis. This ideally identifies the two flanking markers
that show the fewest recombination events with the mutant locus. The first
map provided for the zebrafish anchoring CA-repeats (simple sequencelength
polymorphisms) on the map consisted of 102 markers (Knapik et al.,
1996), but now over 10 000 CA-repeat markers are available (Zebrafish
Webserver, http://zebrafish.mgh.harvard.edu), and more markers are added
onto the map at a regular pace. Testing an additional 10 markers on a panel
of 96 embryos usually will take only a few days. Not all of these markers
may turn out to be polymorphic in the two strains that are being used in a
particular experiment, but in many cases investigators have been able to
limit the interval size to a couple of centimorgans (one centimorgan equals
roughly 660 kbp) or less.
During the third step, the markers defining the interval are used to inititate
a chromosomal walk. Genomic libraries of high quality have been made
available very recently. From every new BAC, PAC or YAC, new SNPs
(single-nucleotide polymorphisms) can be generated and tested for recombination
events. Collecting mutant embryos from a particular strain is not
limiting in fish, and usually more than 2500 embryos (equaling 5000 meioses)
are used for fine mapping, resulting in a resolution of 0.02 cM (or 13 kbp).
Once the interval has been narrowed down sufficiently, the whole region is
sequenced. Sequencing is the fourth step and takes about 4 weeks, depending
on the expertise and the number of sequencing lanes available. From the
genomic sequence, enough coding information can be retrieved to make
predictions about the genes within the region.
The final step is to prove which one of the genes, if mutated, is responsible
for the phenotype. There are a number of ways to accomplish this, and in