Model Organisms in Drug Discovery

GENOMIC TECHNOLOGIES 195
Developing genomic tools such as libraries, meiotic mapping panels and largeinsert
libraries for zebrafish has been slow initially. The work has sped up
considerably, however, with the success of the first two large-scale forward
genetic screens (Driever et al., 1996; Haffter et al., 1996). These groundbreaking
screens succeeded in demonstrating that there were plenty of
mutants to work on, and since that time genomic technologies have advanced
rapidly. The zebrafish genome is about 1.7 Gbp in size, which is a little more
than half the genome size of humans and mice. There are 25 chromosomes
(haploid set) and approximately 2700 cM (Postlethwait et al., 1994, and
references therein).
What steps are involved in cloning a mutant of choice? Basically, there are
five steps: mapping the mutant to a linkage group (chromosome); identifying
flanking markers that define a chromosomal interval in which the mutated
gene is located; generating markers within the interval that allow narrowing
down of the interval size; sequencing the region of interest; and identifying the
gene in question among the coding units within the region. There are detailed
descriptions for how to carry out all of these steps elsewhere (Geisler, 2002,
and references therein), therefore it will suffice here to give a tour-de-raison
through the process, highlighting the existing public resources and pointing
out the time-lines involved in all of these steps.
First, once a mutant has been identified, it is necessary to determine which
chromosome the mutated gene resides on. To that end, a heterozygous carrier
is outcrossed with a wild-type fish from a polymorphic strain, and carriers are
identified from the resulting filial generation. These fish are used to produce
homozygous mutant as well as sibling embryos, both of which are collected
separately. Sorting of homozygous embryos is done phenotypically. The DNA
from both mutant and sibling pools is then used to carry out a number of
PCRs with primers amplifying so-called CA-repeats (microsatellites) – short
DNA fragments that differ in length between polymorphic strains. By
comparing whether particular CA-markers are co-segregating with the
homozygous mutant embryos, it is possible in most cases to establish a
linkage of the mutant gene with one or more of the polymorphic markers. This
candidate linkage is then confirmed by testing individual embryos with such
markers, which confirms and establishes the number of recombination events
between the markers and the mutant locus. Because the PCR products have
been mapped previously, both meiotically and on a radiation hybrid map, the
position of the PCR products is known with respect to the chromosome
(Knapik et al., 1996).
Commonly, a marker set of roughly 200–250 polymorphic markers is used.
Given the genome size of 2700 cM, the average resolution that can be achieved
with this method is of the order of 10 cM. Agarose gels are used to resolve the
polymorphic markers (Geisler, 2002) or, alternatively, acrylamide gels can be
employed, allowing the use of 96-well capillary systems such as the ABI 3700