Target Discovery and Validation Reviews and Protocols

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Transgenic Models 169 a recombinant protein of pharmaceutical interest will be correctly posttranscriptionally modified in transgenic animals. 2.1.5. Use of Episomal Vectors To avoid position effect of chromatin or transgenes, one theoretical possibility consists of using vectors, which remain autonomous as plasmids or minichromosomes. Several viruses, namely, herpes viruses, are maintained for long periods in organisms as independent genomes. Chromosome fragments also can replicate autonomously and be stably maintained. These natural mechanisms are being used to generate autonomous vectors. Vectors containing the origin of replication and Epstein–Barr nuclear antigen-1 gene from Eptein–Barr virus are spontaneously maintained in human cells. Interestingly, plasmids containing a matrix attached region (MAR) can autoreplicate in a number of animal cell types (23). It is not known whether this very flexible vector can be used to generate transgenic animals. Chromosomes fragments containing foreign genes also can be transmitted to daughter cells in vitro (24) and even maintained in transgenic mice (25). Additional studies are required before these tools can be used easily to generate transgenic animals. 2.1.6. Use of Male Gametes Experiments carried out more than one decade ago showed that mouse sperm incubated with DNA could generate transgenic animals after in vivo or in vitro fertilization. This technique proved poorly reproducible in part because sperm contain DNAse, which degrades the foreign DNA (Fig. 2). The technique has been greatly improved, and it allowed the generation of transgenic pigs with a high yield (26,27) and also of transgenic sheep. This approach, which allowed gene transfer in rabbit embryos (28,29), is attractive only for animals in which microinjection is difficult, inefficient, or costly. A new method was defined several years ago to generate transgenic Xenopus. Indeed, DNA microinjection leads to the maintenance of DNA for some stages during embryo development but not to its integration. This method consists of degrading the membrane of isolated sperm to facilitate DNA uptake, incubating this damaged sperm in the presence of DNA, and fertilizing oocytes by using intracytoplasmic sperm injection (ICSI) (30). This method is currently used in Xenopus, and it has been extended to mice (31,32). Interestingly, it was recently shown that long DNA fragments from BAC or yeast artificial chromosome (YAC) vectors could be efficiently transferred to mouse oocytes (32). For unknown reasons, ICSI did not allow the generation of transgenic monkeys (33). Transgenesis in Xenopus has been recently improved after an interesting observation originally done in medaka. DNA fragments released from plasmids by the action of the meganuclease I-Sce 1 (I-Sce) showed a much higher capacity to
170 Houdebine integrate into the medaka genome (34). The mechanism of action of I-Sce 1 is not known. It is postulated that the enzyme remains bound to the DNA fragment, allowing its protection against exonucleases and its unspecific targeting to host DNA. This approach proved successful in Xenopus but not in mice so far. To improve DNA binding to sperm, a group recently used a monoclonal capable of recognizing a surface antigen present in sperm of a number of vertebrates. This antibody contains a basic amino acid-rich region in its C-terminal end, allowing a spontaneous binding of DNA and its transfer to oocytes during in vitro or in vivo fertilization ([35,36]; Fig. 2). Direct DNA transfection into sperm precursors in seminiferous tubules can be achieved in mice (37). Alternatively, sperm precursors may be collected, transfected in vitro, and reimplanted into recipient testis (38–40). These methods are promising but require additional studies before being used. 2.1.7. Use of Pluripotent or Somatic Cells Foreign DNA can be transfected into pluripotent cells or somatic cells further used to generate embryos harboring the transgene. Pluripotent cells, also known as embryonic stem (ES) cells, can be introduced in developing embryos at the morula or the blastocytes stage and give birth to transgenic animals capable of transmitting their transgenes to progeny. In practice, this end can be achieved with only two mouse strains from pluripotent cells that can be cultured. Recent studies showed that ES cells also could be obtained in medaka. Animal cloning by nuclear transfer was rapidly implemented to generate transgenic sheep (41) and cows (42). Although laborious, the method is more efficient than DNA microinjection in ruminant pronuclei, and it has not been adopted for other animals such as rats, rabbits, and pigs. The use of cloning in these species is therefore restricted to targeted gene transfer. 2.2. Targeted DNA Integration For several reasons, it is important to target integration of foreign genes. This approach may avoid uncontrolled position effect of chromatin on transgene expression. This approach is also the major method to specifically inhibit the expression of a cellular gene, also known as gene knockout. 2.2.1. Gene Knockout In all species, although very weakly in plants, a foreign DNA fragment can replace a genomic DNA by a homologous recombination mechanism. This replacement implies that relatively long regions of the endogenous and the foreign DNA have identical sequences (Fig. 3). Homologous recombination is a rare event, and it cannot be achieved at a sufficient rate in one-cell embryos after microinjection of the gene constructs. Gene replacement must therefore be
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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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220 Vijayaraj, Söhl, and Magin abs
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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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