Target Discovery and Validation Reviews and Protocols

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Heart Failure and Immunity 271 We have used a mouse model of postinfarction congestive heart failure combined with various in vitro techniques to specifically study the contribution of cytokines and thereby try to delineate the mechanism whereby heart failure may hamper normal hematopoiesis (11,12). Because we are investigating various aspects of inflammation on heart failure and hematopoiesis, the careful selection and handling of the experimental animals are particularly important to avoidance of undesirable confounding effects. This approach relies on first inducing heart failure by ligation of the left coronary artery to produce an acute myocardial infarction. Heart failure also may be induced by aortic banding, atrial pacing or injection of the right ventricular depressant monocrotaline. After development of overt heart failure, which usually lasts approx 4–6 wk, the animals are sacrificed, and blood and bone marrow samples are collected. Then, in vitro methods are used to pinpoint the contribution of various molecules, e.g. cytokines, to growth and viability of hematopoietic progenitor cells. Importantly, the development of overt heart failure also can be manipulated in vivo by either direct injection of putative regulatory molecules or constant delivery via implanted osmotic minipumps. Here, we present important aspects of this strategy with particular emphasis on adequate handling of the animals and ways of identifying putative inhibitors of hematopoiesis in heart failure mice. 2. Materials 2.1. Experimental Animals For our studies, we have used adult 6- to-8-wk old Balb/c mice (see Note 5). Consent from appropriate ethics committees before commencing the actual experiments is mandatory because these experiments involve induction of a serious and lethal disease. 2.1.1. Health Monitoring Because most infections in laboratory animals are subclinical, the animals and biological material to be used must be tested before the experiments. It is therefore vital to have a sentinel animal program and test regularly for parasitical, bacterial, and viral agents according to the needs of the experiment in question or by following the recommendations of the Federation of European Laboratory Animal Associations (FELASA) (13). 2.1.2. Testing of Biological Material Biological material, such as tumor cell lines, may be tested in antibody production tests, where the biological material is given via different ports of entry (per os, intraperitoneal, intravenous, subcutaneous, and intranasal) to clean animals placed in an isolator. The animals are kept in the isolator for a
272 Iversen and Sørensen Fig. 1. Proposed sequence for studying hematopoiesis in mice with congestive heart failure. minimum of 6 wk before they are euthanized, and serology is used to test for antibodies against the agents in question. The other option is to use PCR to ensure that biological material does not harbor viral contaminants that may influence the outcome of the experiments. It is currently under discussion whether PCR can replace antibody production (14). 2.2. Anesthetics and Postoperative Analgesics Because the induction of myocardial infarction requires vascular surgery on a very small animal (body weight ~25 g), a stable general plane of anesthesia is important. After insertion of an endotracheal tube, we recommend a mixture of 2% isoflurane and 98% oxygen while keeping the animal on a ventilator. During the postoperative phase, repeated (every 8–12 h) subcutaneous injections with buprenorphine (0.3 mg/mL) is used as postoperative analgesia. 3. Methods The experimental protocol is outlined in Fig. 1.
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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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182 Houdebine Fig.7. Example of a v
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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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218 Vijayaraj, Söhl, and Magin sug
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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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