Target Discovery and Validation Reviews and Protocols

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Heart Failure and Immunity 273 3.1. Surgery and Induction of Acute Myocardial Infarction 3.1.1. Ligation of Left Coronary Artery In general anesthesia, a thoracotomy is made, and the left coronary artery is carefully dissected free of the surrounding tissue. Then, a ligature is placed around this artery and tightened (see Note 6). In the sham control animals, this artery is only exposed before the thoracic cavity is closed. 3.2. Secondary Operation and Verification of Heart Failure By experience, a chronic congestive heart failure develops within the next 4 to 6 wk. In general anesthesia, a catheter is introduced into the left ventricular cavity to measure the end-diastolic pressure (LVEDP) (see Note 7). In this model, an elevated LVEDP is a hallmark of heart failure (15). An ultrasound examination of an enlarged left atrium also substantiates a failing heart. Postmortem, the wet-to-dry weight of the lungs is also assessed because this parameter is indicative of congestion (see Note 8). 3.3. Sampling of Blood and Bone Marrow Tissue A blood sample is obtained by heart puncture in deep anesthesia immediately before the mouse is killed. Then, the femoral bone is isolated, and small holes are made in both ends. The femoral marrow is then flushed out of the bone by using a syringe containing either saline or air. 3.4. In Vitro Assays of Hematopoeitic Growth and Leukocyte Activity Hematopoietic cells are cultured on semisolid media (methyl cellulose) for approx 2 wk before colonies are scored, and the numbers obtained are taken as the growth potential of these cells (11). Neutrophilic activity of cells is measured as their oxygen consumption by using an O2-sensitive electrode, whereas lytic activity among lymphocytes (T- and natural killer [NK]-cells) is measured with a conventional 51Cr liberation assay (12). 3.5. Identifying Inhibitors of Hematopoiesis The gene expression of possible inhibitors (e.g., TNFα) is done by an RNase protection assay on candidate cells (T- and NK-cells). The corresponding protein contents are measured with Western blotting (11). 4. Notes 1. The homeotherm animal is able to maintain a constant body temperature, within a range specific to each species, as a response to changes in the surrounding temperature. When subjected to surrounding temperatures outside this range, the body tries to remain in the thermoneutral zone by regulation of the flow of heath between
274 Iversen and Sørensen the core of the body and the body surface. This regulation may affect the metabolic rate of the animal and consequently result in different experimental results compared with an animal in its thermoneutral zone. It is important that the environmental temperature is precisely defined and kept within the defined range. The temperature must be recorded and published. Environmental parameters are listed in Appendix A in the Convention of the Council of Europe. Most rodents may be kept at room temperatures between 20 and 24°C. It is important that the temperature is kept constant, and it must be recorded and published. 2. Relative humidity should be kept in the range of 55±5%. Higher levels of relative humidity may lead to disturbances in the metabolic rate, and excessively high levels of relative humidity predispose for pathological conditions. 3. The light cycle has an important effect on the animal’s physiology and behavior. Although most animals have their periods of highest activity during daylight hours, the majority of laboratory animals are nocturnal and sleep during day. In general 12 h of light and 12 h of dark is recommended for most laboratory animal species. The light intensity must be adjusted to the need of the species and should be constant in all parts of the room. 4. Exposure to chemicals may be a part of the procedure in an experiment or it may be unintentional in the laboratory animal facility. The most important sources of chemical contamination are animal excretions, bedding, cage or enclosure materials, feed, and household chemicals. 5. We prefer to study male mice because these are generally larger than the females. Size is a critical aspect because the induction of myocardial infarction requires surgery on the coronary vessels of the small murine heart. 6. The ligature is tightened so the myocardial tissue is blanched on visual inspection. 7. The use of microtipped transducer catheters is preferable to avoid pressure artifacts. 8. Any weight should be adjusted for possible edema, e.g., by expressing the weight relative to a bone length. References 1. McKee, P. A., Castelli, W. P., McNamara, P. M., and Kannel, W. B. (1971) The natural history of congestive heart failure: the Framingham study. N. Engl. J. Med. 285, 1441–1446. 2. Khand, A., Gemmel, I., Clark, A. L., and Cleland, J. G. (2000) Is the prognosis of heart failure improving? J. Am. Coll. Cardiol. 36, 2284–2286. 3. Kloner, R. A. and Rezkalla, S. H. (2004) Cardiac protection during acute myocardial infarction: where do we stand in 2004. J. Am. Coll. Cardiol. 44, 276–286. 4. Torre-Amione, G., Kapadia, S., Lee, J., et al. (1996) Tumor necrosis factor-alpha and tumor necrosis factor receptors in the failing human heart. Circulation 93, 704–711. 5. Iversen, P. O., Nicolaysen, G., and Sioud, M. (2001) DNA enzyme targeting TNFalpha mRNA improves hemodynamic performance in rats with postinfarction heart failure. Am. J. Physiol. 281, H2211–H2217.
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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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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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216 Vijayaraj, Söhl, and Magin of
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Page 266 and 267: 12 The HUVEC/Matrigel Assay An In V
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Page 290 and 291: 14 An Overview of the Immune System
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Page 332 and 333: 15 Potential Target Antigens for Im
Page 334 and 335: 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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