Target Discovery and Validation Reviews and Protocols

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Transgenic Models 175 Fig. 4. Different elements to be added in a vector to favor transgene expression. Adaptation of the codons to the organism in which the transgene is introduced may greatly enhance its expression. The chemical synthesis of cDNAs or genes is an efficient and not too costly way to obtain these optimized coding regions. Moreover, the 5′ untranslated region (UTR) must contain as little as possible of secondary structure. It is sometimes necessary to generate transgenic animals expressing simultaneously several genes. The simple way consists of coinjecting two independent constructs, which cointegrate in 70–80% of the cases. Alternatively, the two independent constructs may be linked in the same vector. This linkage complicates vector constructions, but it allows a better control of the transgene activity. Another possibility consists of adding sequences known as internal ribosome entry site (IRES) between two cistrons. It is thought that an IRES recruits ribosomes directly, independently of the cap. A study carried out in our laboratory suggests that this mechanism may be not right for all IRESs (61). In practice, it must be known that biscistronic mRNAs containing IRES between the two cistrons express, most of the time, less efficiently their corresponding proteins. This lower expression is particularly true for the second cistron. The cistron to be expressed at the lowest level should preferably be put after the IRES. It also was shown that the second cistron is efficiently expressed if the distance between the termination codon of the first cistron and the beginning of the IRES is of approx 80 nucleotides (61). 3.2. Vectors to Inhibit Specific Gene Expression The understanding of gene function and the generation of animal models rely in part on the specific inhibition of cellular genes. It is also important to inhibit the expression of viral genes to study their function or to generate lines of animals
176 Houdebine Fig. 5. Different methods to inhibit specifically the expression of a gene. (A) Classical gene addition, (B) gene knockout, (C) gene knockdown by targeted gene silencing (TGS), (D) inactivation of a mRNA by hybridizing with an antisense oligonucleotide, (E) knockdown by mRNA inactivation by using siRNA, (F) knockdown by inhibition of mRNA translation by using microRNA, (G) inhibition of a cellular protein by using an intrabody, (H) competitive inhibition of a cellular protein by using overexpression of a transdominant negative protein, and (I) specific destruction of a given cell type in vivo (genomic ablation). resistant to diseases. In Fig. 5, the different levels in which the expression of a gene can be blocked are shown. The levels are essentially genes themselves, mRNAs, and proteins. Indeed, the expected effect is most of the time finally at the protein level. Genes can be specifically inactivated after having been knocked out. In addition, gene function can be inhibited by several mechanisms, such the use of small-interfering RNAs (siRNAs), and synthetic antisense siRNAs and microRNAs can inactivate specifically mRNAs by inducing their degradation or the inhibition of their translation, respectively (see Chapters 4–12, Volume 2). Recombinant intracellular antibodies known as intrabodies can inactivate proteins. An overexpression of transdominant negative proteins playing the role of decoys can abrogate specifically protein function as well. To generate relevant
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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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Page 217 and 218: 204 Vijayaraj, Söhl, and Magin 1.
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226 Vijayaraj, Söhl, and Magin gen
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228 Vijayaraj, Söhl, and Magin the
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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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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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