Model Organisms in Drug Discovery

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ORGANS IN COLOR: TRANSGENIC ZEBRAFISH 193 fragmentation) or when challenged with the anticancer drug camptothecin, zebrafish embryos devoid of p53 exhibited a far lesser degree of apoptosis than control embryos. This experiment shows that p53 function is conserved across species boundaries and, at the same time, that camptothecin acts through p53. The application of chemicals to zebrafish embryos is easily accomplished by bathing the embryos in the respective chemical or, in those cases where penetration turns out to be problematic, injecting the compounds into the embryo. Because this can even be done in a 96-well format (Peterson et al., 2000), scenarios of screening chemicals in zebrafish become feasible. There is one elegant example of this approach in which a cell cycle arrest zebrafish mutant was challenged with thousands of compounds in order to identify successfully the small number of compounds that were able to revert and rescue the mutant phenotype (Len Zon, personal communication). Clearly, the zebrafish has potential as a screening tool and assay system for testing compounds and drugs. How far that potential can reach will, in large part, be determined by the degree of automation that can be integrated into the screening process. 7.5 Organs in color: transgenic zebrafish In addition to forward and reverse genetics, zebrafish offer the opportunity to interfere with gene activity by overexpressing genes, either through injecting in vitro synthesized mRNAs or through transgenesis. The former method applies to genes and processes that have an early effect on development or organ formation. The half-life of the injected mRNA and the corresponding protein determines how late a process can be interfered with. Usually, this is a matter of hours or a couple of days at best. The latter method, transgenesis, is employed in those cases where stable expression of a particular gene is desired, either ubiquitously or in a time- and tissue-specific manner. Transgenes in zebrafish are commonly generated via injection of DNA into the zygote (Gilmour et al., 2002). By a poorly understood process, the DNA is amplified by the embryo and DNA concatamers are integrated at random positions (Stuart et al., 1998). Integration only happens occasionally at the one cell stage, and as a consequence the founder animal (i.e. the fish that initially got injected) more often than not is mosaic, with some cells carrying the transgene and others not. Consequently, it is necessary to test whether the germline of any founder fish carries the transgene. This is accomplished by crossing the founder fish and examining the resulting progeny via PCR or, alternatively, by visual inspection of the animals in cases where a fluorescent gene product results from the transgene. Transgenesis rates in the range 0–20% using this method (Higashijima et al., 1997; Gilmour et al., 2002; Langenau et al., 2003; N.
194 GENETICS AND GENOMICS IN THE ZEBRAFISH Figure 7.1 Angiography of a live zebrafish larva at 3 days of age. Anterior is to the left. Note the high resolution of individual vessels, which are fluorescently labeled Scheer, personal communication). Once established, the transgenic line can be maintained by conventional breeding and the transgene is passed onto the next generations in a strictly Mendelian fashion. It is the transparency of zebrafish that makes using transgenes attractive to researchers. Although transgenic fish have been put to use in a number of cases before, it is the elegant combination of transparency and fluorescently labeled proteins such as green fluorescent protein (GFP) that offers advantages peculiar to the zebrafish (see Figure 7.1). Fluorescent proteins under the control of specific promoters allow the generation of transgenic lines that display fluorescently marked blood (Long et al., 1997), blood vessels (Lawson and Weinstein, 2002) and labeled lymphoid cells (Langenau et al., 2003), to name a few examples. Such lines are useful for cell sorting specific populations but, more importantly, they offer the opportunity to observe biological processes over time in vivo with minimal interference. A beautiful example of this can be viewed under http://dir.nichd.nih.gov/lmg/uvo/ weinslab.html where a rare chance to observe sprouting blood vessels in vivo is offered. Several GFP-labeled lines also have been utilized for screens, where they provide the added advantage of screening the same embryo with more than one assay. For instance, a transgenic line that expresses GFP under the control of a vessel-specific promoter can be analyzed in a screen for mutants lacking vessels, the same embryos can be checked for motility defects a day later and yet another day later they can be fixed and scored for defects in ossification. 7.6 Genomic technologies With all genetic model systems, the development of genomic tools goes hand in hand with genetics, because every interesting phenotype raises an immediate question: which gene has been mutated to cause the phenotypic alteration?
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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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Page 149 and 150: TARGET IDENTIFICATION/TARGET VALIDA
Page 151 and 152: CHEMICAL GENETICS 143 acid identity
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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
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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 193 and 194: 186 GENETICS AND GENOMICS IN THE ZE
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Page 209 and 210: 8 Lipid Metabolism and Signaling in
Page 211 and 212: FISH AS A MODEL ORGANISM 205 genes
Page 213 and 214: LIPID METABOLISM SCREEN 207 transpo
Page 215 and 216: LIPID METABOLISM SCREEN 209 Figure
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Page 219 and 220: ZEBRAFISH AS A MODEL SYSTEM 213 Fig
Page 221 and 222: ZEBRAFISH AS A MODEL SYSTEM 215 Reg
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Page 227 and 228: REFERENCES 221 Patrono, C., Patrign
Page 229 and 230: 224 CHEMICAL MUTAGENESIS IN THE MOU
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246 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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