Target Discovery and Validation Reviews and Protocols

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Transgenic Models 167 2.1.3. Use of Transposons Transposons are mobile DNA fragments that integrate genomes with high efficiency. The gene of interest can replace the transposase gene of the transposons. This replacement prevents the recombinant transposons from being disseminated in an uncontrolled manner. This implies that transposase or a gene coding for this enzyme is coinjected with the transposon to allow its integration. Transposon P is currently used to generate transgenic Drosophila. Different transposons have been designed to be able to transfer genes in a variety of species. The transposon Mariner proved efficient to generate transgenic chickens and medaka. The transposon Sleeping Beauty works in mammals as in medaka (9). In silkworm, a specific transposon, Piggy Bac, is being used successfully (10). These transposons are not significantly complemented by endogenous transposons, and they do not disseminate in host genomes. One of the limitations of this technique is that transposons can harbor only 2 or 3 kilobases (kb) of foreign DNA and that cellular mechanisms recognize these foreign sequences and may silence the transgenes. Research for improving the use of transposons are underway (11,12). 2.1.4. Use of Viral Vectors Retroviral vectors have been extensively studied to become a tool for gene therapy in humans. However, the success with this tool is limited. Yet, retroviral vectors allowed several immunodeficient children to leave their protective bubbles. The efficiency of retroviral vectors has been greatly improved by using lentiviral genomes and vesicular somatitis virus (VSV) envelope (13). Lentiviral vectors have the advantage of transferring their DNA to the nucleus at any stage of the cell cycle. The VSV envelope recognizes any animal cell type, including oocytes and embryos with high efficiency, and it allows the preparation of concentrated viral vectors (Fig. 2). Retroviral vectors were originally used to generate transgenic chickens. Transgenic monkeys also were obtained with conventional retroviral vectors (14). Lentiviral vectors have greatly improved this tool. Transgenic chickens (15), pigs (16), sheep (17), and cows (18) have been generated in this way much more easily than with DNA microinjection or with conventional retroviral vectors (19,20). For unknown reasons, attempts to generate transgene monkeys have not been successful so far (21). Commercial kits are available to generate efficient and safe recombinant lentiviral particles, but care must be taken to manipulate the recombinant particles, especially when the foreign genes are oncogenes or are involved in cell multiplication, apoptosis, or signal transduction to cells. Interestingly, foreign genes transferred by lentiviral vectors are generally well expressed, as opposed to those added to conventional retroviral vectors. This
168 Houdebine Fig. 2. Major methods to introduce genes into oocytes and embryos. high expression may be because lentiviral vectors integrate preferentially in genome regions containing genes and often within transcribed regions. It remains that lentiviral vectors cannot harbor more than 8 or 9 kb of foreign DNA and that the regulatory regions present in the viral long terminal repeat (LTRs) may interfere with the promoters associated to the genes of interest. This interference may reduce the cell specificity of transgene expression. Sophisticated vectors inactivate LTR enhancers, but their manipulation is more complex. In some circumstances, it may be interesting to obtain a transient expression of foreign genes in the whole cells of the organism or in a few cell types only. This end can be achieved by using adenoviral vectors. These vectors are extensively studied for gene therapy in humans. Methods to introduce foreign genes in the adenoviral genome have been standardized. Highly efficiency and safe vectors can be prepared. They remain stable for a few days without being integrated into host genome. Transient expression experiments should determine whether a particular gene has an effect. In a positive effect, classical transgenic animals may be generated to study the function of the transgene. This approach may enhance the chance of creating relevant models. Adenoviral vectors were used to express human growth hormone gene transiently and at a high rate in goat milk (22). In this case, the method seems suitable to predict whether
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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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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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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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