Model Organisms in Drug Discovery

234 CHEMICAL MUTAGENESIS IN THE MOUSE
Figure 9.4 A typical experiment applying gene-driven ENU mutagenesis. The exon
encoding the catalytic loop of Type II cGMP-dependent protein kinase Prgk2 was
amplified by PCR and analysed for point mutations using TGCE. (A) The TGCE profile of
the amplified fragment indicates the presence of a point mutation (upper panel; lower panel
is wild-type control). (B) The point mutation is verified by direct DNA sequencing. (C) The
base pair exchange identified leads to a missense mutation changing lysine (K) to Arginine
(R). (D) The mutation in Prgk2 occurs at an evolutionary highly conserved amino acid
position, as illustrated by the protein sequence alignment. Mm, Mus musculus; Hs, Homo
sapiens; Rn, Rattus norvegicus; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans;
Ag, Anopheles gambiae; Am, Apis mellifera; Bm, Bombyx mori; Oc, Oryctolagus cuniculus;
Bt, Bos taurus; Ho, Hydra oligactis
does not model drug action very well. Rather, alteration of a specific domain
should be the desired change.
Figure 9.4 shows a typical experiment implementing this strategy. In a search
for mutations specifically affecting the enzymatic activity of orphan protein
kinases, a missense mutation leading to a change in an evolutionary highly
conserved amino acid in the catalytic domain of kinase Prkg2 was identified
(Figure 9.4C). The associated sperm sample was used to revitalize the mutant
mouse line, and homozygous offspring are currently under investigation to