Model Organisms in Drug Discovery

REVERSE GENETICS BY ENU MUTAGENESIS 233
gradient HPLC (dHPLC; Coghill et al., 2002) have been applied successfully,
the most promising approach in terms of sensitivity and throughput is
currently temperature gradient capillary electrophoresis (TGCE; Li et al.,
2002).
Using TGCE, the gene of interest is amplified from heterozygous DNA
derived from G1 animals, typically one exon per polymerase chain reaction
(PCR) fragment. If an ENU-induced mutation is present, the PCR product
will contain heteroduplex molecules, showing melting curves in a temperature
gradient different from homoduplexes (Figure 9.4A). Positive fragments are
then sequenced to determine the exact nature of the mutation (silent, missense,
nonsense or splice site; Figure 9.4B).
Recently introduced TGCE machines can sensitively and rapidly identify
heteroduplexes in 2000 or more fragments per day. Assuming a typical gene
with 10 exons, the mutation detection in a substantial G1 repository can be
performed in 1 week or less. Extrapolating from the mutation loads outlined
above, a repository of 1000 samples, equivalent to onefold genome coverage,
would provide a 60% statistical chance of obtaining one loss-of-function allele
in any given gene of interest. With a repository size of 5000, the likelihood of
identifying one allele would be 495%, with a 70% chance to obtain two
alleles (Coghill et al., 2002). A repository size of 10 000 might be desirable and
will be within the range of mutation detection technology.
Applications of gene-driven ENU mutagenesis
Reverse genetics by ENU is very scalable because it requires only minimal
human input in comparison to the specific design and construction of
recombinant vectors necessary for gene targeting. The availability of the
mouse genomic sequence facilitates the semi-automatic design of primers for
DNA amplification, and rapid mutation detection technologies such as TGCE
are highly amenable to industrialization.
Its main application is in the physiological validation of candidate drug
targets. The rapid and cost-effective availability of informative genetic
variation in target genes in various genetic backgrounds lowers the threshold
to the use of the mouse model as early as possible in a drug discovery
program. In particular, the investigation of orphan druggable genes will profit
from application of the technology, because targeted knock-outs frequently
only uncover the first step in development where the mutated gene is
absolutely required, whereas point mutations frequently provide additional
information due to hypomorphic changes or partial loss of function.
Point mutations can mimic drug action more closely than gene deletions.
Because drugs usually do not work by eliminating the target protein, but by
inducing specific changes in function, removal of the protein in a null allele