Target Discovery and Validation Reviews and Protocols

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Keratin Transgenics and Knockouts 231 6. 20 mL 2X Freezing medium: mix 10 mL of complete ES medium, 4 mL of ultrapure dimethyl sulfoxide, and 6 mL of FCS. Store at –20°C. 7. 10X HEPES-buffered saline (HBS) for electroporation: mix 16 g of NaCl, 0.74 g of KCl, 0.252 g of Na 2 HPO 4 , 2 g of D-glucose (dextrose), and 10 g of HEPES. Dissolve all in 180 mL of cell culture grade water, adjust pH to 7.2, bring up to 200 mL, and then filter through 0.1-µm pore filter. Store at –20°C. To prepare 1X HBS, dilute with sterile water, which brings pH to 7.05. HAT and HT media are bought as 50X stocks from Sigma and used as if they were 100X concentrated. To prepare 100X HAT, dissolve hypoxanthine in 2 mL of 1 N NaOH, add 8 mL of cell culture grade water, and mix well. Add another 10 mL of water and mix until most of the hypoxanthine is dissolved. Add 1 mL of 1 N NaOH and mix until hypoxanthin is dissolved. Subsequently, add 75 mL of water, thymidine, and then 100 mL of water. Add aminopterin and mix again (aminopterin is a poison, so consult material safety data sheet). Filter through 0.1-µm filter and then store as 20-mL aliquots at –20°C. 8. 1 mM Aminopterin: weigh out 100 mg and dissolve in 45 mL of cell culture grade water. Add about 1 mL of 1 N NaOH to dissolve aminopterin, sterilize as in item 7, and then store at –20°C. 9. Additional reagents: a. 1X Trypsin inhibitor from Glycine max (soybean; cat. no. 074K1366, Sigma). Store as 10-mL aliquots at –20°C. b. 200X Pen/strep (Invitrogen) and stored as 1-mL aliquots at –20°C. c. G418 (Invitrogen or any other manufacturer). Prepare 40 mg/mL in PBS and filter sterilize. Store at –20°C. Final concentration used is 350–400 µg/mL. d. Puromycin dihydrochloride (Calbiochem). Reconstituted in water to get a stock concentration of 3 mg/mL. Final concentration used is 3 µg/mL. e. Hygromycin B solution (Calbiochem). The final concentration used is 150 µg/mL. f. Primocin (cat. no. Amaxa Biosystems) and used against mycoplasms as recommended by supplier. When using primocin, Pen/Strep is not additionally required in the medium. g. 6-TG is prepared as a 1000X stock (i.e., 2 mM). Dissolve 50 mg in small volume of 1 N NaOH. Add water up to 25 mL, dissolve completely, and filter sterilize. Store as 5-mL aliquots at –20°C. h. LIF is prepared according to protocol. i. 0.3 M Mannitol (cat. no. M4125, SIgma) is dissolved in ultrapure water with 0.3% BSA added (cat. no. A4378, Sigma); filter through 0.22-µm Millipore filter and store in aliquots at –20°C. 2.2. Websites, Tables, Vendors, and Vectors 2.2.1. Online Databases 1. http://www.mshri.on.ca/nagy/Cre-pub.html. 2. http://jaxmice.jax.org/models/cre_intro.html.
232 Vijayaraj, Söhl, and Magin 3. http://genetics.med.harvard.edu/~dymecki/. 4. http://www.ensembl.org/Mus_musculus/. 2.2.2. Tables Conditional alleles in mice: tissue-specific knockouts, kkwan@mail. mdanderson.org. 2.2.3. Vendors 1. http://www.invitrogen.com/. 2. http://www.sigmaaldrich.com. 3. http://www.rndsystems.com/. 4. www.calbiochem.com. 2.2.4. Vectors 1. http://www.aldevron.com/vectors.php?vid=5. 2. http://www.stratagene.com/lit/vector.aspx. 3. http://www.genome.gov/10001852. 4. Mutant Lox P vectors, hiroshi@genetics.hpi-uni-hamburg.de. 3. Methods 3.1. Routine Culture, Generation, and Characterization of Genetically Altered Mouse ES Cells Here, we describe culture of HM-1 cells, which are derived from the mouse strain 129/Ola and are HPRT-deficient. They are grown feeder-free and in gelatinized dishes. Other feeder-independent ES cell lines are grown essentially in the same way. For routine culture, cells should be passaged twice a week, splitting them between 1:6 and 1:12, depending on conditions. They should not be plated too thin because thin plating encourages excessive differentiation. Overconfluent cultures should be avoided for the same reason. Medium change should take place every other day as long as the culture is thin; later, medium should be changed every day. Any drastic change in cell doubling times or cell morphology is indicative of unwanted damage to the cells, e.g., chromosomal aberrations (see Note 2). Typical doubling time is approx 20–22 h (see Fig. 5). 3.2. Thawing and Plating Cells 1. Gelatinize flask by using enough gelatin to completely cover the flask or dish. 2. Leave at room temperature (RT) for at least 10 min. Aspirate off before plating cells. 3. If feeders are used, plate appropriate amount of cells together with ES cells or plate them 3 to 4 h earlier. Feeder-coated dishes can be used up to 7–10 d. Check for integrity of monolayer before using them to plate ES cells. 4. Thaw cells quickly in 37°C bath and transfer into 10-mL prewarmed medium. Spin for 5 min at 250g.
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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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Page 217 and 218: 204 Vijayaraj, Söhl, and Magin 1.
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Page 290 and 291: 14 An Overview of the Immune System
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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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