Target Discovery and Validation Reviews and Protocols

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Keratin Transgenics and Knockouts 217 was established after mating these mice with a strain expressing a truncated progesterone receptor-1–Cre-recombinase fusion protein under the control of regulatory elements of either the K5 or K14 gene promoter (73). Upon induction with antiprogestin or RU486 only in those cells expressing either K5 or K14, the Cre-recombinase will be induced to excise the floxed neo-cassette inserted after the first exon of the mutated K14 transgene (72), thus activating the transgene. Correspondingly, blisters filled with fluid on the front legs and paws became visible but healed after 2 wk. The unexpected healing process was explained with the partial activation of the Cre-recombinase in the nonstem cell population of basal keratinocytes. The migration and reepithelialization by nonphenotypic stem cells (containing the floxed neo-cassette) or transiently amplifying cells derived from these stem cells of the untreated surrounding areas was finally considered as the working hypothesis (72). 1.1.5.3. SUPRABASAL STRATIFIED EPITHELIA In the upper epidermis, 60% of the total protein content consists of keratins K1 and K10 (74). Surprisingly, epidermal integrity was maintained in neonates after targeted disruption of K10 (Fig. 3G). Adult K10(–/–) animals showed a fivefold increase of basal cell proliferation and hyperkeratosis (75) In the suprabasal cell layer of the neonatal skin, an IF network composed of K1, K5, and K14 keratins compensates for the loss of K10 (Fig. 3G), whereas within the adult skin, an induced upregulation of the cytokeratins K6a, K6b, K16, and K17 might take over at least some of the functions of K10-containing intermediate filaments (75). Moreover, an induction of cyclin D1 and of c-myc in basal cells as well as that of the cell cycle regulator 14-3-3 in postmitotic keratinocytes was noted (76), implying an involvement of K10 in the regulation of epidermal cell proliferation (77). Cell transfection experiments indicated the K10 head domain to sequester both the Akt as well as the atypical protein kinase Cζ kinases avoiding their translocation to the plasma membrane, thus representing a negative regulator of epidermal cell cycle (78). Therefore, K10 was proposed to suppress cell proliferation; however, the K10(–/–) strain contradicts this view because a general increase of suprabasal cell proliferation could not be observed (79) except for an increase in sebocyte proliferation and differentiation. Sebaceous gland cells of K10(–/–) mice showed an accelerated turnover and secreted more sebum, including wax esters, triglycerides, and cholesterol esters. It was concluded that the suprabasal intermediate filament cytoskeleton devoid of keratin K10 increased the differentiation of epidermal stem cells toward the sebocyte lineage (79). Furthermore, K10(–/–) mice treated with 7,12dimethylbenz[a]anthracene/12-O-tetradecanoylphorbol-13-acetate developed far less papillomas than wild-type mice. 5-Bromo-2′-deoxyuridine-labeling revealed a strongly accelerated keratinocyte turnover in K10(–/–) epidermis,
218 Vijayaraj, Söhl, and Magin suggesting an increased elimination of initiated keratinocytes at early stages of developing tumors. The concomitant increase in K6a, K16, and K17 in K10deficient epidermis and the increased motility of keratinocytes is in agreement with the pliability versus resilience hypothesis, proclaiming that K10 and K1 render cells more stable and static. The K10(–/–) knockout represent the first mouse model showing that loss of a cytoskeletal keratin reduces tumor formation. This response might be achieved by an accelerated turnover of keratinocytes, possibly mediated by activation of mitogen-activated protein kinase pathways (80). Expression of a truncated and dominant negative form of K10 (81,82) produced skin lesions comparable with severe forms of epidermolytic hyperkeratosis (EHK) (see Fig. 3C). Moreover, homozygous mice (K10T/K10T) revealed changes in the composition of epidermal lipids and barrier function (83,84). Transgenic mice, generated as described for K14 (72), harboring a K10 point mutation (R154C, equivalent to R156C in the human), produced local blisters and scaling at the sites of induction that persisted later in life together with unaffected epidermis. Apparently, epidermal stem cells have been targeted in these mice. In agreement with mosaic form of EHK in humans, which is because of postzygotic somatic mutations, this finding shows that in the absence of selective pressure—the mutant K10 is silent in presumptive stem cells—mutant and wild-type stem cells can coexist. This idea has to be considered in therapy approaches of EHK; they must include either ablation or correction of mutant stem cells (85). Generally, extensively bundled IFs consisting of K1/K10 parallel the surface of flatted keratinocytes, being covalently cross-linked to cornfield envelope (CE) proteins (86). However, K10 mutations, resulting in EHK, do not affect CE integrity (87), which is in contrast at least to some mutations found in K1. K1 frameshift mutations resulting in the shortening of its glycine-rich V2 tail domain generate the dominant inherited skin disorder ichthyosis hysterix (88,89), most likely caused by the inability of the occurring IFs to form bundles. Loricrin, but not involucrin and filaggrin, was found irregularly distributed throughout the cytoplasm of keratinocytes, thus pointing to a specific role of the K1–loricrin interaction during CE formation (90). Although the targeted ablation of keratin K1 is not published yet (Meier-Bornheim et al., in preparation), an interesting aspect to clarify might be if the distribution or assembly of proteins directed to the CE is also disturbed after the loss of K1 within IFs. The ectodermal dysplasia called pachyonychia congenita (PC) affects epithelial appendages and results in profoundly dyskeratotic nails (Fig. 3E). It can be divided into type 1 (Jadahsson–Lewandowski) PC characterized by oral leukoplakia and palmar–plantar keratoderma and type 2 (Jackson–Lawler) PC typified by subepidermal cysts, natal teeth, but generally milder nail lesions (91). Type 1 PC is
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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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Transfected Cell Arrays 159 2. The
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Transfected Cell Arrays 161 5. Simp
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164 Houdebine Fig. 1. Different met
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Page 266 and 267: 12 The HUVEC/Matrigel Assay An In V
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268 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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