Target Discovery and Validation Reviews and Protocols

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Keratin Transgenics and Knockouts 209 or constitutively K1, K2e, K9, and K10 in mouse and human (39–41). More related to epidermal appendages, the novel keratin 17 (K17n) gene is mainly expressed in nail tissue as well as in fili- and fungiform papillae of the oral mucosa (36). Nevertheless, the aforementioned concept of “keratin expression pairs” is proven by the exception that there is no mouse ortholog to the human K3 gene (3). In the differentiated layers of the human cornea, both K3 and K12 are abundantly expressed; the former keratin has to be replaced by K5 in the mouse (42). During embryonic development, keratin protein expression starts very early with K8 and K18 at ED2.5 (32) being detectable in the surface ectoderm (43,44) and is followed by K5 (ED9.25) and K14 (ED9.75) (32). Shortly afterward, at ED11.5, keratins K6a, K6b, K16, and K17 appear, even before the onset of stratification at approx ED13.5 in the mouse (45). In this respect, it is intriguing that the mouse K14 gene might be a direct target of the p53 tumor suppressor homolog p63 because K14 was expressed at very low levels in the single layer of p63-deficient epidermis of mouse (46). 1.1.4. Keratin Function in Cell Signaling and Keratinopathies Mutations within keratin genes lead to various epidermal diseases. The diseases have different pathologies mainly caused by cell fragility, which could be accompanied by a disturbance of the normal cell signaling (1,47,48). An electronic database including various keratin mutations is available: at www.interfil.org. The most prominent K14R125C mutation not only leads to EBS (Fig. 3A) because of keratin (IF) aggregate formation coinciding with decreased resilience to mechanical forces but also to a weakening of the K14–tumor necrosis factor (TNF)α receptor-associated death domain (TRADD). This weakening leaves transfected keratinocytes more susceptible for caspase-8–mediated apoptosis caused by an alleviated cytoprotection mediated by K14–TRADD interactions (49). Furthermore, cell culture studies with these transfected keratinocytes revealed that keratin aggregates are in a dynamic equilibrium with soluble subunits, suggesting that dominant negative mutations act by altering cytoskeletal dynamics and solubility largely depending on the “mutant to wild-type” ratio (19). Keratin mutations render keratinocytes more susceptible to osmotic shock and activate the stress-activated protein kinase/c-Jun NH 2 -terminal kinase (JNK) pathway (50). Generally, point mutations in or truncation of epidermal keratins result in visible monogenic keratinopathies, whereas phenotypes of mutated embryonic and internal epithelial keratins are much more subtle. Depending on the population, K8 as well as K18 gene mutations (see Tables 1 and 2) have been associated with cryptogenic cirrhosis, liver disorders, and chronic pancreatitis as well as
210 Table 1 Orthologous Human and Mouse Keratin Genes of Type I Cluster Human Mouse Major Knockout Human keratin keratin expression phenotype pathology Reference K9 K9 n.d. Unknown K10 K10 Suprabasal Hyperkeratosis, Non-epidermolytic 75,77 epidermis acanthosis; K14 hyperkeratosis compensation K11P n.d. Unknown K12 K12 Cornea Cell fragility Meesmann’s Ko et al., 1996 corneal (see Fig. 3B) dystrophy K13 K13 n.d. Unknown K14 K14 Basal Postnatal death Epidermolysis 35 epidermis at P4, skin bullosa simplex (see Fig. 3A) blistering (EBS) cytolysis of basal cells K15 K15 n.d. Unknown K16 K16 n.d. Pachyonychia 93 congenita (see Fig. 3E)
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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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144 Fig. 1. The ribozyme-expression
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146 Sano and Taira 5. 10X L (low-sa
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148 Sano and Taira ribozymes may cl
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150 Fig. 3. A system for the identi
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152 Sano and Taira 4. Notes 1. We r
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9 Production of siRNA- and cDNA-Tra
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Transfected Cell Arrays 157 Fig. 1.
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260 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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