Target Discovery and Validation Reviews and Protocols

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Keratin Transgenics and Knockouts 243 4. Notes 1. It is important to keep ES cell passage number as low as possible to maximize germline competence and to minimize genetic and epigenetic alterations. It is recommended to prepare a sufficiently large stock of frozen vials and to expand a vial every time for a new experiment. In general, ES cells should be split at rates between 1:6 and 1:12. High- and low-density plating carries the risk of differentiating the cells. Their growth rate (typical doubling time is 20–22 h) should be carefully monitored; significant changes in growth rate and morphological appearance signal chromosomal alterations and preclude further use of such cells for gene-targeting experiments. 2. To retain their excellent germline transmission potential, it is essential to keep passage number of HM-1 cells as low as possible. Good germline transmission rates depend on (1) media, plasticware, and so on; (2) limiting passage number; and (3) experimental skills. We use cell culture plastic from BD Biosciences, cell culture grade water, and reagents. 3. More details about human and mouse keratin clusters can be found in ref. 3. Acknowledgments We thank Ursula Reuter, Silke Loch, and Claudia Wohlenberg for excellent technical assistance. Work by the authors was supported by the Deutsche Forschungsgemeinschaft, the Bundesministerium für Bildung und Forschung, and the Thyssen foundation. We apologize to those colleagues whose work has not been cited because of space restrictions. References 1. Omary, M. B., Coulombe, P. A., and McLean, W. H. (2004) Intermediate filament proteins and their associated diseases. N Engl J Med 351, 2087–2100. 2. Hesse, M., Magin, T. M., and Weber, K. (2001) Genes for intermediate filament proteins and the draft sequence of the human genome: novel keratin genes and a surprisingly high number of pseudogenes related to keratin genes 8 and 18. J. Cell Sci. 114, 2569–2575. 3. Hesse, M., Zimek, A., Weber, K., and Magin, T. M. (2004) Comprehensive analysis of keratin gene clusters in humans and rodents. Eur. J. Cell. Biol. 83, 19–26. 4. Magin, T. M., Reichelt, J., and J. C. (2005) The role of keratins in epithelial homeostasis, Elias review, submitted. 5. Rogers, M. A., Langbein, L., Winter, H., et al. (2001) Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded in the type I keratin gene domain on chromosome 17q12-21. J. Biol. Chem. 276, 19,440–19,451. 6. Bawden, C. S., McLaughlan, C., Nesci, A., and Rogers, G. (2001) A unique type I keratin intermediate filament gene family is abundantly expressed in the inner root sheaths of sheep and human hair follicles. J. Invest. Dermatol. 116, 157–166.
244 Vijayaraj, Söhl, and Magin 7. Zimek, A. and Weber, K. (2005) Terrestrial vertebrates have two keratin gene clusters; striking differences in teleost fish. Eur. J. Cell Biol. 84, 623–635. 8. Herrmann, H., Hesse, M., Reichenzeller, M., Aebi, U., and Magin, T. M. (2003) Functional complexity of intermediate filament cytoskeletons: from structure to assembly to gene ablation. Int. Rev. Cytol. 223, 83–175. 9. Herrmann, H., Strelkov, S. V., Feja, B., et al. (2000) The intermediate filament protein consensus motif of helix 2B: its atomic structure and contribution to assembly. J. Mol. Biol. 298, 817–832. 10. Herrmann, H., Wedig, T., Porter, R. M., Lane, E. B., and Aebi, U. (2002) Characterization of early assembly intermediates of recombinant human keratins. J. Struct. Biol. 137, 82–96. 11. Strelkov, S. V., Herrmann, H., Geisler, N., et al. (2002) Conserved segments 1A and 2B of the intermediate filament dimer: their atomic structures and role in filament assembly. EMBO J. 21, 1255–1266. 12. Herrmann, H. and Aebi, U. (2004) Intermediate filaments: molecular structure, assembly mechanism, and integration into functionally distinct intracellular Scaffolds. Annu. Rev. Biochem. 73, 749–789. 13. Irvine, A. D. and McLean, W. H. (1999) Human keratin diseases: the increasing spectrum of disease and subtlety of the phenotype-genotype correlation. Br. J. Dermatol. 140, 815–828. 14. Whittock, N. V., Ashton, G. H., Griffiths, W. A., Eady, R. A., and McGrath, J. A. (2001) New mutations in keratin 1 that cause bullous congenital ichthyosiform erythroderma and keratin 2e that cause ichthyosis bullosa of Siemens. Br. J. Dermatol. 145, 330–335. 15. Whittock, N. V., Wan, H., Morley, S. M., et al. (2002) Compound heterozygosity for non-sense and mis-sense mutations in desmoplakin underlies skin fragility/woolly hair syndrome. J. Invest. Dermatol. 118, 232–238. 16. Coulombe, P. A., Hutton, M. E., Letai, A., Hebert, A., Paller, A. S., and Fuchs, E. (1991) Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses. Cell 66, 1301–1311. 17. Lane, E. B., Rugg, E. L., Navsaria, H., et al. (1992) A mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering. Nature 356, 244–246. 18. Ma, L., Yamada, S., Wirtz, D., and Coulombe, P. A. (2001) A ‘hot-spot’ mutation alters the mechanical properties of keratin filament networks. Nat. Cell Biol. 3, 503–506. 19. Werner, N. S., Windoffer, R., Strnad, P., Grund, C., Leube, R. E., and Magin, T. M. (2004) Epidermolysis bullosa simplex-type mutations alter the dynamics of the keratin cytoskeleton and reveal a contribution of actin to the transport of keratin subunits. Mol. Biol. Cell 15, 990–1002. 20. Windoffer, R., Woll, S., Strnad, P., and Leube, R. E. (2004) Identification of novel principles of keratin filament network turnover in living cells. Mol. Biol. Cell 15, 2436–2448. 21. Peters, B., Kirfel, J., Bussow, H., Vidal, M., and Magin, T. M. (2001) Complete cytolysis and neonatal lethality in keratin 5 knockout mice reveal its fundamental
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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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166 Houdebine concentrations become
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168 Houdebine Fig. 2. Major methods
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170 Houdebine integrate into the me
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172 Houdebine 2.2.3. Knock-In Into
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174 Houdebine as insulators. The co
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176 Houdebine Fig. 5. Different met
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178 Houdebine systems. Moreover, st
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180 Houdebine contribute to generat
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182 Houdebine Fig.7. Example of a v
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184 Houdebine more extensively. Tra
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186 Houdebine and stroma. Several o
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188 Houdebine explained at the mole
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190 Houdebine of the intestinal epi
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Overview of Immune System 293 (unre
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Overview of Immune System 295 Fig.
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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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