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Mutant candidates or phenodeviants are identified by comparison with control
mice of the same genetic background. It is therefore critical to prepare a reliable and
large-scale control phenotype dataset. Phenodeviants are mated to produce the G2
offspring, which is called the inheritance test cross. If a candidate phenotype anom-
aly occurred due to an ENU-induced mutation, the phenotype should be observed in
half of the G2 offspring. Only the phenodeviants that passed the inheritance test are
registered as mutants.
Mutants have been identified in approximately two to three percent of G1 mice
using the basic screening platforms of RIKEN and other ENU mouse mutagenesis
projects. Thus, the expected number of dominant mutant lines that have been newly
generated in the past 5 years by large-scale ENU mouse mutagenesis programs is
roughly 5000 (= 2 - 3% x 20,000 G1 x 10 projects). About the same number of mu-
tant lines have been identified and established during the 100-year history of mouse
genetics. Transgenics and gene targeting have produced an equivalent number of
mutants since the late 1980s. Altogether, more than 15,000 mutant lines are available
as a resource for studying gene and genome functions.
10.3.2.2 Phase II: Gene Identification and Recessive Screens
Connecting the causative genomic DNA changes to the phenotype outcome demands
the identification of the ENU-induced mutation site that is responsible for the estab-
lished lines. The classical genetics approach of backcrossing, to map the mutation in
the mouse chromosomes or genome-based candidate approaches are combined to
identify the site of the mutation. Currently, both mapping and positional cloning are
utilized to locate the mutations obtained by ENU mouse mutagenesis programs.
Another key objective of Phase II of the large-scale ENU mouse mutagenesis
project is to systematically establish recessive mutants. While many dominant mu-
tants have been collected, many human genetic diseases are recessive. Dominant
screens have also revealed that some genes exhibit their dominant phenotype due to
loss of function. In such cases, other genes that would show the same phenotypes by
gain of function can hardly be identified. The mutation rate of dominant mutations
due to the loss of function is estimated to be about 10 -3 /locus/G1. This frequency is
probably 1000-fold higher than the frequency for gain of function. In order to estab-
lish comprehensive human genetic disease models that cover most of the mouse
genes, it is necessary to develop recessive screens. As shown in Fig. 6, recessive
screens take much more time, space, and manpower.
10.3.2.3 Informatics Infrastructure for ENU Mouse Mutagenesis
Database System . Large-scale ENU mouse mutagenesis requires the construction
and organization of an informatics infrastructure. RIKEN chose Oracle database as
the core of the infrastructure (Gondo 2001; Masuya et al. 2004). All manipulations,
protocols, data recordings, data analyses, annotations, etc. are stored in and retrieved
from the database server by multiple client machines. Some examples are described
below.
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