Model Organisms in Drug Discovery

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138 DROSOPHILA – A MODEL SYSTEM Figure 5.4 Single-nucleotide polymorphism (SNP) mapping of a gene * with two alleles * 1 and * 2 . The * 1 /* 2 combination is lethal. Two marker P elements on both sides of the mutation are used for recombination. Single recombinant flies are tested for their recombination profile. The two closest SNPs on either side of the mutation, SNP2 and SNP3, respectively, which are shown to be homozygous in at least one recombinant, define the critical region for the mutation mutation from each side represent the maximal interval in which the mutation is located. In this way, the mutation in question can be mapped to a few tens of kilobase pairs. From the genes annotated in this region, the gene carrying the mutation is identified again by DHPLC. The DNA is extracted from flies heterozygous for different alleles and the original chromosomes used in the mutagenesis. Fragments uncovering the coding regions of the candidate genes are amplified and examined for an altered elution profile. Such a profile is indicative of a sequence difference between the mutant and the original chromosome. The mutations then have to be verified by DNA sequencing.
TARGET IDENTIFICATION/TARGET VALIDATION STRATEGIES 139 Reverse genetic approaches Reverse genetic approaches are important to investigate the function of genes, of which only the sequence and maybe the expression profile is known. In organisms less amenable to forward genetic approaches, such as the mouse, reverse genetics is the predominant approach. The techniques developed in the mouse to inactivate genes by homologous recombination have revolutionized this model system for basic biological research and for drug discovery (Capecchi, 1989). Genes related to human diseases can be efficiently inactivated in the mouse and suitable disease models often ensue. Although gene targeting by homologous recombination is well established in mice and in yeast, this technique has been introduced only recently in Drosophila (Rong and Golic, 2000; Rong et al., 2002). Are these techniques even necessary in model organisms such as Drosophila, given their powerful forward genetic tool kit discussed above? It is the rather negative answer to this question that may explain why it took so long for these techniques to be developed. We argue, however, that reverse genetic approaches are important also in Drosophila. With the completion of the human genome, there are increasing numbers of genes whose function needs to be studied and their products need to be assigned to particular biological pathways. Of more than 1000 genes associated with human diseases, more than 70% are conserved in Drosophila (Reiter et al., 2001). For many of these, the function is not known in humans or in Drosophila. Having a LOF mutation in such a gene is a starting point (and not the final goal) of a functional analysis. The mutant phenotype cannot be analyzed in isolation. By testing for genetic interactions with other mutations, it may be possible to assign the corresponding gene product to a given signaling pathway and/or biological process. For this genetic characterization, which follows the initial discovery of the mutant phenotype, the Drosophila model offers strong advantages over more complex systems such as the mouse: short generation time, large collection of mutants and sensitized signaling pathways, to name just a few. Therefore, for Drosophila as a model in target discovery and target validation, reverse genetic approaches are important. In the following, we will discuss the advances in two of these techniques in Drosophila: gene targeting and post-transcriptional gene silencing using RNA interference (RNAi). Targeted gene disruption With the first successful gene targeting by homologous recombination 2 years ago, one of the important drawbacks in Drosophila research was eliminated. A general and efficient method to target basically every gene is now available (Rong and Golic, 2000; Rong et al., 2002). Targeted gene disruption is
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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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84 DROSOPHILA AS A TOOL FOR DRUG DI
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Page 109 and 110: 100 DROSOPHILA AS A TOOL FOR DRUG D
Page 127 and 128: 5 Drosophila - a Model System for T
Page 129 and 130: EVOLUTIONARY CONSERVATION OF DISEAS
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Page 151 and 152: CHEMICAL GENETICS 143 acid identity
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Page 155 and 156: about their function in a multicell
Page 157 and 158: REFERENCES 149 Han, Z. S., Enslen,
Page 159 and 160: REFERENCES 151 Rintelen, F., Stocke
Page 161 and 162: 6 Mechanism of Action in Model Orga
Page 163 and 164: INTRODUCTION TO COMPOUND DEVELOPMEN
Page 165 and 166: MODEL ORGANISMS ARRIVE ON THE SCENE
Page 167 and 168: ELUCIDATING THE MECHANISM OF COMPOU
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Page 171 and 172: A CASE STUDY FOR ALZHEIMER’S DISE
Page 179 and 180: NEW CHEMICAL GENETIC STRATEGIES 171
Page 181 and 182: A CASE STUDY FOR INNATE IMMUNITY 17
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Page 187 and 188: REFERENCES 179 Austin, J. and Kimbl
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Page 191 and 192: REFERENCES 183 Sin, N., Meng, L., W
Page 193 and 194: 186 GENETICS AND GENOMICS IN THE ZE
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190 GENETICS AND GENOMICS IN THE ZE
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8 Lipid Metabolism and Signaling in
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FISH AS A MODEL ORGANISM 205 genes
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LIPID METABOLISM SCREEN 207 transpo
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LIPID METABOLISM SCREEN 209 Figure
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the embryo media containing radioac
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ZEBRAFISH AS A MODEL SYSTEM 213 Fig
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ZEBRAFISH AS A MODEL SYSTEM 215 Reg
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aspirin, which has potent inhibitor
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REFERENCES 219 Chau, I. and Cunning
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REFERENCES 221 Patrono, C., Patrign
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224 CHEMICAL MUTAGENESIS IN THE MOU
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252 SATURATION SCREENING OF DRUGGAB
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280 INDEX bupropion 92 busulfan 143
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282 INDEX Dscam 171 dual-energy X-r
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284 INDEX L-685,818 16 lead discove
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286 INDEX protein function 19-22 pr
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288 INDEX yeast (continued) functio
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