Target Discovery and Validation Reviews and Protocols

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Keratin Transgenics and Knockouts 241 2. Wash aggregation needle briefly in ethanol. 3. Make about six aggregation wells into each of the drops by pressing and turning the needle into the ground of the tissue culture dish. 4. Place aggregation plate in an incubator at 37°C, 5%CO 2 for at least 2 h. 3.11.5. Removal of Zona Pellucida With Tyrode’s Solution The following supplemental materials are needed for these experiments: dissecting microscope, acid Tyrode’s solution (cat. no. T1788, Sigma), tissue culture dishes (100 × 15 mm), M2 and KSOM-AA medium, and mouth pipet. 1. Add embryos to the first M2 drop. Rinse 20 embryos in the first drop of Tyrode’s solution and transfer them into the second drop. 2. Observe dissolving of the zona pellucida with the microscope and instantly transfer the embryos to the M2 drop containing 0.3% BSA to prevent embryos from sticking to the plate or to each other. 3. Wash embryos by transferring them through 3 drops of M2 and 3 drops of KSOM-AA. 4. Transfer embryos into a KSOM-AA drop without depressions on the aggregation plate. 5. Trypsinize ES cells for aggregation if ES cell/tetraploid embryo aggregation is planned. 3.11.6. ES Cells or Diploid Embryo/Tetraploid Embryo “Sandwich” Aggregation (Table 3) The following supplemental materials are needed for these experiments: dissecting microscope, prepared aggregation plate with depressions and embryos with removed zona, trypsinized ES cells or eight-cell embryos, and mouthcontrolled pipet. 1. Transfer tetraploid embryos in 1 microdrop of the aggregation plate without depressions. 2. Also transfer diploid eight-cell knockout embryos or trypsinized ES cells into another microdrop. 3. Place a tetraploid embryo in each of the aggregation wells. 4. Place an eight-cell embryo or a clump of approx 10 ES cells on the tetraploid embryo in each depression. 5. Place a second tetraploid embryo on top of the eight-cell embryo or ES cell clumps, respectively, thus forming a sandwich. 6. Repeat steps 2 to 4 for all aggregations and culture the plate overnight in an incubator at 37°C and 5% CO 2 . 7. On the afternoon of the next day check aggregates for blastocyst formation. Transfer blastocysts into the uteri of day 2.5 p.c. pseudopregnant foster females.
242 Table 3 Time Schedule For Aggregation of Tetraploid–Diploid Embryos Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Superovulation Superovulation Check for Flushing Flushing of Transfer of CD-1 and 5 U of hCG copulation of two-cell eight-cell into uterus knockout plugs embryos embryos of day 2.5 pseudopregnant recipients Electrofusion Setting up aggregation 5 U of PMSG Setting up Mating of Setting up matings of recipients aggregation CD-1 and plate knockout Incubation o/n, 1:1 5% CO 2 at 37°C Incubation o/n, 5% CO 2 at 37°C
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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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Overview of Immune System 291 amino
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Overview of Immune System 293 (unre
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Overview of Immune System 295 Fig.
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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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