Target Discovery and Validation Reviews and Protocols

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Overview of Immune System 287 Once a functional heavy chain is made, it associates with other proteins into a signaling complex known as the pre-BCR (Fig. 4). In addition to a homodimer of Igµ, the pre-BCR contains the products of the surrogate light chain (SLC) genes λ5 and VpreB and a heterodimeric signaling module made up of the transmembrane proteins Igα and Igβ (CD79α and CD79β) (17,24). A developing pro-B-cell that makes a functional Ig heavy chain is rescued from apoptosis and enters the pre-B-cell compartment, whereas nonproductive VDJH rearrangement on both alleles leads to programmed cell death (25). Cell surface expression of a functional pre-BCR induces two to five cell divisions, leading to a large pre-B-cell population that subsequently becomes small resting pre-B-cells (24,25). Notably, this process generates B-cell clones expressing a common µ heavy chain but with the potential to express a diverse array of light chains, thereby extending B-cell repertoire. Similarly to T-cells, the pre-BCR functions as a biological sensor that informs pro-B-cells that they have succeeded in rearranging a functional Ig heavy chain (17). Once a functional/productive µ heavy chain is formed, the cell proceeds to the pre-B-stage, expresses the recombinase, and renders the light chain loci (λ and κ) accessible to the recombination machinery. The κ-chain locus is generally rearranged before the λ-chain locus; the latter chain only initiates rearrangement if the κ-locus rearrangement has failed to generate a productive light chain. In contrast to T-cells, the mechanism of allelic exclusion also occurs at the light chain loci. As soon as a functional light chain has paired with the preexisting µ-heavy chain, a complete IgM molecule is displayed on the cell surface in association with Igα and Igβ, forming the BCR of immature B-cell (Fig. 4). If the newly expressed receptor encounters a strong interaction with self-antigens, B-cell development is inhibited, and the cell is deleted by apoptosis. This process is the first negative selection process that B-cells undergo in the bone marrow. Negative selection at this stage would therefore promote elimination of unwanted autoreactive specificities while preserving immature B-cells reactive with foreign antigens. However, a large proportion of autoreactive B-cells escape clonal deletion by altering their BCRs specificity, so that they are no longer autoreactive, a process called “receptor editing” (26,27) (Fig. 4). Thus, immature B-cells maintain the rearrangement machinery upregulated to allow for secondary rearrangements at the IgL chain gene loci. In addition to clonal deletion, autoreactive B-cells are inactivated via a second mechanism called anergy (25). Anergic B-cells express a unique surface phenotype in that they express much reduced sIgM (because of receptor modulation after antigenic exposure), normal IgD levels, and are desensitized to further receptor-induced signal transduction. The “choice” of an immature B-cells to undergo either clonal deletion or clonal anergy is mainly determined by the concentration
288 Sioud Fig. 5. Affinity maturation of B-cells within the germinal center. Antigen recognized by the surface IgM of the B-cells is internalized, processed, and presented on major histocompatability complex (MHC) class II molecule of the B-cells. The antigen can then be presented to a primed specific T-cell. The T-cells in turn produce cytokines, resulting in B-cell division and maturation to antibody-secreting cells. This activation occurs mainly within the germinal centers of lymph nodes, where B-cells proliferate in the dark zone as centroblasts. These cells do not express surface Ig (Ig–). During cell division they can accumulate V (D)J hypermutation. Subsequently, some cells express membrane antibodies with various affinities and migrate into the germinal center light zone as centrocytes with surface Ig (Ig+). Within the light zone, the centrocytes recover the exogenous antigens from follicular dendritic cells. Antibody–antigen complexes are bound to complement and Fc receptors on the follicular dendritic cell surface. Antigens that bind with high affinity to the antibody will lead to the positive selection of the centrocytes, resulting into the progression into plasma and memory B-cells. Antigens that bind weakly to the antibody would either activate RAG proteins and rearrangement at the Ig light chain (IgL) locus (receptor revision) or induce apoptosis in B-cells. Revised receptors may acquire high antigenic affinity, resulting in B-cell export to the periphery and further maturation. and valency of the interacting self-antigens. In general, highly expressed membrane-bound antigens or antigens with multiple identical epitopes induce extensive BCR cross-linking, leading to immature B deletion. In contrast,
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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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192 Houdebine interference of the g
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194 Houdebine 17. Whitelaw, C. B. A
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196 Houdebine 51. Bell, A. C., West
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198 Houdebine 86. Carmichael, G. G.
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200 Houdebine 121. Pailhoux, E., Vi
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202 Houdebine 156. Auerbach, A. B.,
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204 Vijayaraj, Söhl, and Magin 1.
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206 Vijayaraj, Söhl, and Magin Fig
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208 Vijayaraj, Söhl, and Magin fil
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210 Table 1 Orthologous Human and M
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212 Table 2 Orthologous Human and M
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214 Vijayaraj, Söhl, and Magin ulc
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222 Vijayaraj, Söhl, and Magin 1.2
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224 Vijayaraj, Söhl, and Magin Fig
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226 Vijayaraj, Söhl, and Magin gen
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230 Vijayaraj, Söhl, and Magin f.
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232 Vijayaraj, Söhl, and Magin 3.
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234 Vijayaraj, Söhl, and Magin b.
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Page 334 and 335: Tumor Antigen Discovery 321 develop
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Page 340 and 341: 16 Identification of Tumor Antigens
Page 342 and 343: Immunoproteomics 329 by “shot gun
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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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