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Fig. 7.
Overall scheme of the ENU-based gene-driven mutagenesis at RIKEN. All the G1 males
(see Fig. 5) are subjected to sperm cryopreservation and genomic DNA extraction to construct
“Frozen sperm archive” and “Genomic DNA archive,” respectively. To construct mutant mouse
carrying a point mutation in the target gene, several appropriate PCR primer pairs are designed.
The target gene is amplified and screened for any ENU-induced mutations from the genomic
DNA archive. Once a mutation is found, the corresponding sperm sample in the frozen sperm
archive is used for
in vitro
fertilization (IVF) to reconstruct the strain as live mice.
target gene. On the other hand, if the focus is on the protein-coding sequences of the
target genes, PCR primer design and screening are simpler and faster. Most cDNA
sequence information is deposited in public databases. In addition, cDNA sequences
are much more compact and shorter than genomic sequences. Nevertheless, the suc-
cess of cDNA-based screening depends on the careful selection of tissues that ex-
press the target mRNA and the sampling time.
At RIKEN, genomic DNA was chosen to construct the mutant DNA archive
when we started the system development in 2000. We assumed that the mouse ge-
nome project would be completed and all the mouse genomic sequences would
become available within a few years. Indeed, 2 years later the Mouse Genome Se-
quencing Consortium (2002) published the first paper. The updated mouse genomic
sequences are available from the Ensembl database (http://www.ensembl.org/).
We started a feasibility study with a new mutation discovery system by using a direct
sequencing method, when the number of G1 in the archive exceeded 2,000. Although we
