Model Organisms in Drug Discovery

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INDUSTRIALIZED POSITIONAL CLONING 245 informative animals derived from a mapping cross are available (see below). With high-quality genomic sequences available for humans, mice and rats, the gene content of a candidate region can be analyzed rapidly in silico and compared with syntenic regions of other species for detailed annotation of potential coding regions and conserved non-coding sequences. Candidate genes in the region can be prioritized by previously existing information about their function, as well as by information about the expression pattern derived from expressed sequence tag databases or microarray experiments. The final step – the testing of candidate genes for mutations – is limited only by the cost of DNA sequencing and alternative mutation detection technologies. A typical positional cloning project can be finished in well under a year. For a fully penetrant recessive phenotype the generation of informative meiotic recombinations will be achieved in a simple outcross–intercross breeding strategy (Silver, 1995), that takes approximately 6 months. With modern genotyping technology, mapping the mutated locus to a resolution of 1–2 cM can be achieved in less than 1 month, once the F2 animals are phenotyped. Thus, the limiting factor for positional cloning of ENU mutants is not the application of high-throughput genomics technologies, but the specific characteristics of the mutant under investigation. Phenotypes affecting breeding performance will delay the generation of informative meioses, and quantitative phenotypes modified by genetic background make it more challenging to classify affected and unaffected animals. For example, A/J mice are highly susceptible to allergen-induced airway hyperresponsiveness (AHR), an asthma-related phenotype, whereas C3H/HeJ and C57BL/6 are much more resistant (De Sanctis et al., 1995; Ewart et al., 2000; Karp et al., 2000). Similarly, A/J mice respond much more than C57BL/6 in an immediate cutaneous hypersensitivity test, which is a test for atopy (Daser et al., 2000). Such strain differences strongly affect the strategy for positional cloning of the mutation; also, the mutant phenotype needs to be expressed in the mixed background. Outcrossing a C3H/HeJ mutant that is resistant to allergeninduced AHR with A/J mice, which have a dominant allergen-resistant AHR phenotype (De Sanctis et al., 1995), could lead to loss of the phenotype. Finally, once the mutation hunt in the candidate region is ongoing, it is obvious that coding mutations in known genes already linked to the physiology of interest can be identified very rapidly, whereas mutations in novel ‘orphan’ genes or non-coding mutations require more downstream work to be proved beyond doubt as the cause of the mutant phenotype. A recent report (Herron et al., 2002) describes the rapid mapping of 7/15 ENU-induced recessive developmental mutations using interval haplotype analysis and the identification of the causative mutation in two of these lines. From the first phase of the ENU mutagenesis screen at Ingenium, 30 mutations were cloned in a time-frame of 15 months and 20 additional loci
246 CHEMICAL MUTAGENESIS IN THE MOUSE were mapped (manuscript in preparation), demonstrating that the positional cloning process can be established at an industrial scale. Table 9.2 shows the distribution of the molecular characteristics (missense, nonsense, splice site, non-coding) of the mutations characterized so far. No regional bias to specific chromosomes or chromosome segments was observed. 9.7 Conclusions and Prospects The rediscovery and large-scale application of random mutagenesis using ENU is an important extension of the mouse genetics toolkit, which can be straightforwardly integrated into existing drug discovery strategies. When applied in gene-driven reverse genetics experiments, it has been established as a scalable alternative to ES-cell based mutagenesis technologies in target validation. As a tool for the discovery of new therapeutic principles, it provides the opportunity to investigate new physiological pathways by means of phenotype-driven forward genetics screens, which are unrestricted by existing knowledge of molecular entry points. The improved availability of mouse models will facilitate their systematic application in pharmaceutical research. 9.8 References Table 9.2 Summary of the molecular changes identified by positional cloning in the Ingenium ENU screen Mutation type 1 n 2 % Missense 15 56% Nonsense 3 11% Splice site 7 26% Non-coding 2 7% AT/TA transversion 11 41% AT/GC transition 8 30% CG/AT transition 7 26% GC/CG transversion 1 4% Total 27 100% 1Type of change in gene structure and DNA sequence, respectively. 2Number of mutant mouse lines analyzed. Alessandrini, F., Jakob, T., Wolf, A., Wolf, E., Balling, R., Hrabe, de Angolis, M. H., Ring, J., et al. (2001). Enu mouse mutagenesis: generation of mouse mutants with aberrant plasma IgE levels. Int. Arch. Allergy Immunol. 124, 25–28.
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Model Organisms in Drug Discovery M
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Copyright u 2003 John Wiley & Sons
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Contents List of contributors .....
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CONTENTS ix 7 Genetics and Genomics
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List of Contributors Hector Beltran
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LIST OF CONTRIBUTORS xiii Stefan Sc
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1 Introduction to Model Systems in
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Organism INTEGRATING MODEL ORGANISM
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INTEGRATING MODEL ORGANISM RESEARCH
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INTEGRATING MODEL ORGANISM RESEARCH
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10 GROWING YEAST FOR FUN AND PROFIT
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3 Caenorhabditis elegans Functional
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THE DRUG DISCOVERY PROCESS 43 thoug
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multivulva phenotype of Ras gain-of
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disease. These molecular targets ar
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FROM DISEASE TO TARGET 49 Figure 3.
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FROM DISEASE TO TARGET 51 Figure 3.
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signaling pathway. The third catego
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FROM DISEASE TO TARGET 55 resistant
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phenomenon was first observed in C.
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profiles. The C. elegans gene expre
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emerging technologies. Forward gene
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The compound library LEAD DISCOVERY
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LEAD DISCOVERY 65 previous section,
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LEAD DISCOVERY 67 Figure 3.5 Distri
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Compound learning set for assay val
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10 000-15 000 human drug targets ar
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transporter itself. The SSRIs that
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REFERENCES 75 de Montigny, C., Blie
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REFERENCES 77 Lewis, J. A., Wu, C.
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REFERENCES 79 Tissenbaum, H. A. and
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82 DROSOPHILA AS A TOOL FOR DRUG DI
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100 DROSOPHILA AS A TOOL FOR DRUG D
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5 Drosophila - a Model System for T
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EVOLUTIONARY CONSERVATION OF DISEAS
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TARGET IDENTIFICATION/TARGET VALIDA
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CHEMICAL GENETICS 143 acid identity
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3. A hit identifies a biologically
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about their function in a multicell
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REFERENCES 149 Han, Z. S., Enslen,
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REFERENCES 151 Rintelen, F., Stocke
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6 Mechanism of Action in Model Orga
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INTRODUCTION TO COMPOUND DEVELOPMEN
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MODEL ORGANISMS ARRIVE ON THE SCENE
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ELUCIDATING THE MECHANISM OF COMPOU
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ELUCIDATING THE MECHANISM OF COMPOU
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A CASE STUDY FOR ALZHEIMER’S DISE
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NEW CHEMICAL GENETIC STRATEGIES 171
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A CASE STUDY FOR INNATE IMMUNITY 17
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GLOBAL GENE EXPRESSION STUDIES IN M
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As described above, the extensive i
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REFERENCES 179 Austin, J. and Kimbl
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REFERENCES 181 Hughes, T. R., Marto
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REFERENCES 183 Sin, N., Meng, L., W
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186 GENETICS AND GENOMICS IN THE ZE
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Page 199 and 200: 192 GENETICS AND GENOMICS IN THE ZE
Page 209 and 210: 8 Lipid Metabolism and Signaling in
Page 211 and 212: FISH AS A MODEL ORGANISM 205 genes
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Page 257 and 258: 252 SATURATION SCREENING OF DRUGGAB
Page 285 and 286: 280 INDEX bupropion 92 busulfan 143
Page 287 and 288: 282 INDEX Dscam 171 dual-energy X-r
Page 289 and 290: 284 INDEX L-685,818 16 lead discove
Page 291 and 292: 286 INDEX protein function 19-22 pr
Page 293: 288 INDEX yeast (continued) functio
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