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reason we take sperm from all the G1 males when they are 12 weeks old and subject
them to cryopreservation before the onset time.
The frozen sperm archive is considered to be the mutant mouse library. A key
question is how many mutations are indeed preserved in this archive. We started to
estimate the ENU-induced mutation rate per base pair in since 2001 and found
roughly one mutation per million base pairs per gamete (Sakuraba, Sezutsu,
Takahasi, Tsuchihashi, Ichikawa, Fujimoto, Kaneko, Nakai, Uchiyama, Goda,
Motoi, Ikeda, Karashima, Inoue, Kaneda, Masuya, Minowa, Noguchi, Toyoda,
Sakaki, Wakana, Noda, Shiroishi, and Gondo 2005). Recently, results of other
groups supported our findings (Quwailid, Hugill, Dear, Vizor, Wells, Horner, Fuller,
Weedon, McMath, Woodman, Edwards, Campbell, Rodger, Carey, Roberts, Glenister,
Lalanne, Parkinson, Coghill, McKeone, Cox, Willan, Greenfield, Keays, Brady,
Spurr, Gray, Hunter, Brown, and Cox 2004; Augustin, Sedlmeier, Peters, Huffstadt,
Kochmann, Simon, Schöniger, Garke-Mayerthaler, Laufs, Mayhaus, Franke, Klose,
Graupner, Kurzmann, Zinser, Wolf, Voelkel, Kellner, Kilian, Seelig, Koppius,
Teubner, Korthaus, Nehls, and Wattler 2005; Michaud, Culiat, Klebig, Barker, Cain,
Carpenter, Easter, Foster, Gardner, Guo, Houser, Hughes, Kerley, Liu, Olszewski,
Pinn, Shaw, Shinpock, Wymore, Rinchik, and Johnson 2005). Based on this muta-
tion rate, each G1 is expected to carry 3000 ENU-induced heterozygous mutations in
3 × 10
9
bp of the paternally inherited genome. So far we have archived about 10,000
G1 (Sakuraba
et al. 2005) male sperm samples at RIKEN. Worldwide there are
more than 40,000 G1 sperm archives. Within several years the total number of G1
sperm archives will exceed 100,000. The cumulative number of ENU-induced muta-
tions will be
3,000 mutations
∕
G1 × 100,000 G1 = 3 × 10
8
mutations (1)
or one mutation per 10 bp on average. This level is considered to be semisaturated. If
the size of the target gene is 10,000 bp, 100 independent mutations in the gene would
be available in the frozen sperm archive. This number of stored mutants should be
more than sufficient to elucidate the molecular and biological function(s) of a gene in
detail.
10.3.3.2 New Point Mutation Discovery System
If we consider for practical reasons the frozen sperm archive equivalent to the mutant
mouse library, the next key question is how can we discover effectively ENU-
induced point mutations in target genes. The basic scheme to identify ENU-induced
mutation in target genes is depicted in Fig. 7. First, PCR primer pairs are designed to
amplify the sequences of the target gene. Mutations can be identified in the PCR
products by direct sequencing. For practical reasons, it is necessary to prepare either
genomic DNA or cDNA archives from all G1 males. If we screen the genomic se-
quences of the target genes, genomic DNA is simply isolated from any organs or
tissues of the G1 males. However, all genomic sequence information including the
exon and intron structure must be known to permit the design of PCR primers for the
