Target Discovery and Validation Reviews and Protocols

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Transgenic Models 185 be fixed and are thus dead before proceeding to the enzymatic reaction. This death prevents repeated observation on the same organ. However, the β-galactosidase gene is GC rich and frequently silenced in transgenic animals. A redesigned β-galactosidase gene in which most of the codons rich in GC sequences have been replaced by isocodons is available. It does not induce gene silencing. Genes for green fluorescent protein (GFP) and other related proteins having different colors under UV light are more and more frequently used. They offer the advantage of being easily and repeatedly observed in the same tissues without using invasive techniques if the organ is in the periphery of the body (118,119). Moreover, the observation does not require cell death. Interestingly, a device, Cell Vizio, has been created to observe GFP-labeled cells individually in living animals (119a). Transgenic rabbits expressing GFP in all their cells (120) are currently used to follow cells after grafting of organs to other rabbits. Because of its size, the rabbit makes this approach easier than in mice. Essentially, in all the major animal species in which transgenesis has been achieved, GFP animals have been generated to be used as models. One limitation of GFP may be that this protein becomes cytotoxic at high concentration (120). Other specific examples are also worth consideration. Homozygous goat lines without horns are subfertile. It has been shown that this infertility is because of a mutation responsible for an abnormal differentiation of fetal gonads (121). This abnormality is similar to some human genetic diseases. Mice are not relevant models for this study (122). Transgenic goats obtained by cloning are currently being used for this purpose (E. Pailhoux, personal communication, 2005). Several hormone receptor genes have been overexpressed or knocked out to determine their role in tissue growth and differentiation. The mammary gland is composed of two major categories of cells. The parenchyma contains the secretory epithelial and the myoepithelial cells. The stroma, which surrounds the parenchyma, is formed by fibroblasts, adipocytes, and connective tissue. Progesterone is one of the hormones favoring mammary gland growth in vivo and preventing milk secretion during pregnancy until parturition. The progesterone receptor gene is particularly present in the parenchyma. To determine its role, the progesterone receptor gene was knocked out. The development of the mammary gland was impaired in these animals. The grafting of the fetal parenchyma from knocked out mice into the stroma of normal mice did not lead to a development of the mammary gland. Conversely, normal parenchyma grafted into the stroma of knocked out mice developed to become a normal mammary gland. These experiments confirm the role of progesterone in mammary gland development, and they point out the different roles of parenchyma
186 Houdebine and stroma. Several other gene modifications in mice have been carried out to decipher the different mechanisms in the development and the differentiation of the mammary gland (123). 4.4. Models for Cystic Fibrosis Cystic fibrosis results from mutations of the CFTR gene. However, several hundred mutations have been found in this gene, but only some of them cause a severe pathogenicity. In contrast, it is known that the most potent mutations, namely, the ∆F 508 mutation, have not the same effect in all patients. Thus, the cooperation with other genes may enhance or attenuate the effect of the mutation. Transgenic mice harboring the ∆F 508 mutation in their CFTR gene have been independently generated by several groups. In no single case did the models seem fully relevant. CFTR is a chloride channel that is less active and not properly transported to plasma membrane in mutants. Mice have other chloride channels, and their lungs are only weakly affected by the CFTR gene knockout. Yet, these animals show intestinal disorders typical of cystic fibrosis. Rabbit and sheep are expected to be better models for cystic fibrosis because their CFTR genes are more similar to the human CFTR gene. Experiments are underway to generate rabbits and sheep with the mutated CFTR gene, but the major hurdle is that the cloning technique by nuclear transfer must be implemented. The siRNA approach might be an alternative requiring less laborious techniques. However, inactivation of mRNAs by siRNAs is often not complete, and the residual expression of mouse CFTR might diminish the relevance of the model. 4.5. Models Requiring Several Transgenes In many cases, a mutated gene increases the probability of a disease developing but is insufficient alone. This situation is seen with the breast cancer BRCA1 and BRCA2 genes in human breast cancer. Several transgenes must then be expressed simultaneously to generate the pathology. The cooperative effect of two genes is sometimes unexpected. The transgenic mice used to study amyotrophic lateral sclerosis exemplify this point. The crossing of two lines of mice, one harboring the superoxide dismutase-1 gene and the other gene for a neurofilament subunit gave much more relevant models than each line separately (124). Occasionally, the effect of a knockout gene may be compensated by the action of a very different gene, revealing a nonanticipated role of the second gene. Mice in which the LAM2 gene has been knocked out suffer from congenital muscular dystrophy. The addition of the agrin gene known to be involved in the formation of neuromuscular junctions restored muscular function (125). Alzheimer’s disease (AD) is complex, and it occurs after a long
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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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Page 191 and 192: 178 Houdebine systems. Moreover, st
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Page 217 and 218: 204 Vijayaraj, Söhl, and Magin 1.
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236 Vijayaraj, Söhl, and Magin 4.
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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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