Model Organisms in Drug Discovery

REVERSE GENETICS BY ENU MUTAGENESIS 231
confounded by the interaction of mutations. A closer look at the numbers
described above indicates that this is extraordinarily unlikely; 30–35
mutations in a recombinational genome size of 1453 cM (Silver, 1995) amount
to an average genetic distance between two functionally relevant mutations of
41.5–48.4 cM, indicating that adjacent mutations are almost certain to
segregate in the next generation. The average distance of base pair exchanges
of 1–2.5 per Mb is large enough so that for every functional mutation even the
neighbouring silent can be segregated in a simple cross.
Because usually experimentation on a given mutant line will not be done on
the founder animal, but in G2 and subsequent generations, the appropriate
breeding strategy for the maintenance of a mutant will be enough to provide a
clean genetic background. A similar routine backcrossing scheme is good
practice also in embryonic stem (ES) cell-based experiments to eliminate
unlinked spontaneous mutations that might have arisen during cell culture.
9.3 Reverse genetics by ENU mutagenesis
The application of ENU mutagenesis in reverse genetics, i.e. gene-driven
strategies, is very straightforward: from a pool of carrier animals, mutations in
a gene of interest can be identified rapidly and mouse lines carrying the desired
mutations can be established (Coghill et al., 2002) (Figure 9.3).
Compared with standard gene-driven mutagenesis approaches, such as gene
targeting in ES cells, this strategy offers several advantages. An allelic series of
point mutations, including hypomorph or domain specific changes, can be
generated without extra effort. Because the mutant mouse line is established
from frozen sperm samples (Figures 9.3B and 9.3C) rather than ES cells, both
male and female carrier animals are available in the first generation, allowing
direct intercrossing for the generation of homozygotes (Figure 9.3D). This
cuts out the typical ES cell chimera stage and thus shortens the experimental
schedule by one breeding generation, i.e. at least 3 months. Last, but not least,
ENU mutagenesis is not restricted to certain genetic backgrounds, whereas ES
cells are usually derived from the ‘129’ family or from hybrid backgrounds.
On the downside, gene-driven ENU mutants do not allow the specific
design of desired mutations to the extent possible in ES cells. Also, conditional
mutants are not possible. It is most likely, therefore, that the ENU approach
will complement but not supersede ES cell technology.
In practice, this approach requires a repository of G1 animals representing
one or preferably several genome coverages. Rather than maintaining this
repository as a constantly renewing pool of living animals, the genetic
diversity is typically conserved by sperm freezing, with the establishment of a
parallel repository of somatic DNA to be used in mutation screening