Target Discovery and Validation Reviews and Protocols

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Transgenic Models 187 period of incubation. AD is characterized by the extracellular accumulation of polymerized tau protein. β-Amyloid is cleaved from amyloid precursor protein (ADP), the cleavage being performed by three secretases: -α, -β, and -γ (126). Transgenic mice, in which the ADP gene had been knocked out, kept their sensitivity to the disease (127) because of the presence of another gene called ADP-like gene. The knockout of the gene is lethal. Transgenic mice with knocked-out ApoE and IL1 genes are less sensitive to the disease, indicating that these two genes contribute to AD development. Different models have been obtained. Some of the models overexpress normal APD and prenisilin genes, whereas other models express mutated forms of the genes. None of these models reflect all the aspects of AD. Fewer neurons in generals are destroyed in the models than in patients. Correlation between behavioral disorders and accumulation of β-amyloid is not always strict. Yet, the models show a similar disease pattern, and they may therefore help to identify active molecules and to design analogs capable of reaching the brain (128). 4.6. Models for Degenerative Diseases Degenerative diseases generally have slow development, and transgenic models may be helpful to decipher their mechanisms, not only for Alzheimer’s disease but also for prion diseases and other pathologies. Prion diseases would be much less well understood without the contribution of transgenic mice (129,130). Creutzfeldt–Jakob and related diseases are typically pathologies, which incubate for very long periods and require the cooperation of several cell types, especially when infection occurs by the oral route. It was essential to show that the PrP–/– mice were insensitive to any prion infection, which then demonstrated that the PrP gene has a major role in prion diseases. Transgenic mice overexpressing the PrP genes from different species become more sensitive to the prion disease of the same species. This phenomenon is amplified when the endogenous mouse PrP gene is knocked out. For example, PrP–/– mice expressing the bovine PrP gene are sensitive to bovine spongiform encephalopathy (131). PrP–/– mice expressing the hamster PrP gene specifically in astrocytes are sensitive to hamster scrapie (132). Transgenic mice also revealed that some epitopes of the PrP protein are essential for the transmission of the disease (133), that some PrP alleles are more favorable for injection than others, and that genetic environmental factors modify the incubation period of bovine spongiform encephalopathy (134). The prion disease manifests more rapidly when the PrP gene of the mouse and the cell extract used for infection come from more closely related species. It was also surprising to observe that the cell extracts become increasingly infectious after repeated passages to animals. This adaptation has not yet been
188 Houdebine explained at the molecular level. All these studies in vivo have led to the definition of more flexible in vitro systems in which cultured cells can transform the Prp c into PrP sc (135). Various transgenic mice are invaluable models for the evaluation of the therapeutic effects of chemical compounds. Encouraging results showing that different drugs can delay or even partly cure scrapie have recently been obtained. Other brain degenerative diseases are being studied by using transgenic models. Transgenic mice, which overexpress the α-synuclein gene in their brain, are used to study Parkinson’s disease (136). Huntington’s disease also is studied in transgenic mice overexpressing the Huntington gene variants having oligoglutamines of variable length in their N terminus (137). Apoptosis plays a major role in many biological events and is highly conserved in evolution. It is indispensable for development and organogenesis that imply specific cell elimination. Defaults in apoptosis may lead to autoimmune diseases, tumor formation, and neurodegeneracy. At least 25 genes involved in apoptosis have been knocked out (138), including genes for caspases, adaptors, regulators, bcl2 family numbers, and mitochondrial proteins. Aging is a complex phenomenon that has only been partially described. Defects in genomes seem to be a major cause of aging. A growing number of transgenic models are being used to study aging. Mice in which the XPD gene has been knocked out are more sensitive to oxidative DNA damage. This sensitivity was increased further when the XPA gene also was knocked out (139). These models reflect some of the agent syndromes in human. Arthrosclerosis results from complex disorders in lipid metabolism. Many genes are involved in this process. The disease has a genetic component, and its development is favored by cholesterol-rich diets. Numerous genes coding for different apolipoproteins, lipases, and other factors have been added or knocked out in mice (140). These studies have given important information as to the role of the different genes. However, lipid metabolism is very different in mouse and human. Mice must be submitted to diets very rich in cholesterol to mimic even partially atherosclerosis. The rabbit is much closer to humans for this biological function, and this animal is used extensively in addition to mice for the study of atherosclerosis (141). Some transgenic rabbits also are used to define new drugs capable of protecting humans against arteriosclerosis. Cancer occurs under multiple forms, and it is the result of several steps occurring sequentially. All these steps result from alterations of oncogenes and antioncogene expression and activity. Mutations, chromosomal translocations, and environmental factors are thus responsible for tumor formation. Transgenic mice were soon used to try to generate models for cancer study. The first oncomouse expressed c-myc gene in the mammary gland. Further studies made it possible to identify additional genes involved in mammary cancer
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METHODS IN MOLECULAR BIOLOGY 360 T
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M E T H O D S I N M O L E C U L A R
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© 2007 Humana Press Inc. 999 River
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vi Preface get. Approaches to targe
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viii Contents 10 Transgenic Animal
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x Contributors SAHOHIME MATSUMOTO
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xii Contents of Volume 2 12 Validat
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2 Sioud disease pathogenesis and pe
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4 Sioud A more fruitful approach fo
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6 Sioud both transient and permanen
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8 Sioud serum biomarkers. This syne
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10 Sioud Fig. 2. Schematic of targe
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12 Sioud 13. Magin, T. M. (1998) Le
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Harnessing the Human Genome 15 The
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Harnessing the Human Genome 17 Fig.
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Harnessing the Human Genome 23 The
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Harnessing the Human Genome 27 repr
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Harnessing the Human Genome 29 14.
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Harnessing the Human Genome 31 45.
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34 Imoto et al. (4,5), and Bayesian
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36 Imoto et al. 2. Materials Two ty
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38 Imoto et al. Fig. 3. Graphical v
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40 Imoto et al. Fig. 5. Result of t
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42 Imoto et al. p(D |G) = ∫ p(D,
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44 Imoto et al. β k (k = 1,…,q j
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46 Imoto et al. Fig. 7. Partial res
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48 Imoto et al. Fig. 8. Strategy to
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50 Imoto et al. Fig. 9. (continued)
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52 Imoto et al. Table 3 List of Roo
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54 Imoto et al. 5. De Hoon, M. J. L
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56 Imoto et al. 39. Kamimura, T., S
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58 Beaty et al. cytotoxic drugs and
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60 Beaty et al. Fig. 1. Principles
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62 Beaty et al. oligonucleotide mic
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64 Beaty et al. 3. The 12% polyacry
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66 Beaty et al. 2.3.3. Protein Stai
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68 Beaty et al. 3.1.3. Labeling of
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70 Beaty et al. 1 µL of the anneal
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72 Beaty et al. 4. Phenol:cholorfor
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74 Beaty et al. 19. Wash twice with
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76 Beaty et al. The Following Volum
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78 Beaty et al. using a protein ass
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80 Beaty et al. 6. Shake the gel fo
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82 Beaty et al. Fig. 2. Preparation
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84 Beaty et al. search in. A widely
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86 Beaty et al. 3. Kern, S. E., Hru
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88 Beaty et al. 34. Peters, D. G.,
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5 Molecular Classification of Breas
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Molecular Profiling of Breast Cance
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Fig. 2. (Continued) the right ident
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6 Discovery of Differentially Expre
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Gene Target Discovery 117 In the fi
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Gene Target Discovery 119 sequences
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Gene Target Discovery 121 There is
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Gene Target Discovery 123 digestion
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Gene Target Discovery 125 Fig. 1. P
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Gene Target Discovery 127 5. Sargen
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Gene Target Discovery 129 41. Berry
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132 Matsumoto, Miyagishi, and Taira
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134 Matsumoto, Miyagishi, and Taira
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Page 217 and 218: 204 Vijayaraj, Söhl, and Magin 1.
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238 Vijayaraj, Söhl, and Magin 1.
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240 Vijayaraj, Söhl, and Magin alt
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242 Table 3 Time Schedule For Aggre
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250 Vijayaraj, Söhl, and Magin 101
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12 The HUVEC/Matrigel Assay An In V
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256 Skovseth, Küchler, and Haralds
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270 Iversen and Sørensen witnessed
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272 Iversen and Sørensen Fig. 1. P
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274 Iversen and Sørensen the core
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14 An Overview of the Immune System
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Overview of Immune System 279 diffe
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Overview of Immune System 281 Fig.
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Overview of Immune System 283 then
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Overview of Immune System 285 expre
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Overview of Immune System 287 Once
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Overview of Immune System 289 solub
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Overview of Immune System 291 amino
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Overview of Immune System 293 (unre
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Overview of Immune System 297 the c
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Overview of Immune System 299 (44).
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Overview of Immune System 301 demon
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Overview of Immune System 307 some
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Overview of Immune System 309 sera
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Overview of Immune System 313 measu
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Overview of Immune System 315 42. H
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Overview of Immune System 317 74. D
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15 Potential Target Antigens for Im
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Tumor Antigen Discovery 321 develop
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Tumor Antigen Discovery 323 panel b
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Tumor Antigen Discovery 325 16. Joc
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16 Identification of Tumor Antigens
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Immunoproteomics 329 by “shot gun
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Immunoproteomics 331 isoelectric fo
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Immunoproteomics 333 3. 2D-PAGE is
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17 Protein Arrays A Versatile Toolb
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Protein Arrays 337 Table 1 Classifi
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Protein Arrays 339 fractions from c
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Protein Arrays 341 combinatorial sc
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Protein Arrays 343 The ideal proteo
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Protein Arrays 345 18. Cekaite, H.
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Protein Arrays 347 49. Yuko Kawahas
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Index 349 Index A Acetyl-CoA carbox
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Index 351 conditional by using FRT
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Index 353 Recombinant tumor antigen
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