Target Discovery and Validation Reviews and Protocols

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Keratin Transgenics and Knockouts 223 the latter case, HPRT-deficient ES cells are required. Only ES cells that harbor an HPRT-minigene can grow in hypoxanthine, aminopterin, and thymidine (HAT)-containing selective medium. The major advantage of the HPRT system is that it allows for negative selection in medium containing 6-thioguanine (6-TG) (103). In our experience, we have not observed any advantage for the use of negative selectable marker genes outside the homology arms. In most cases, selectable marker genes with their own promoters, usually PGK-1, are used. In rare instances, the phosphoglycerate kinase (PGK)-1 promoter becomes silenced because it carries a partial pCpG island and may have to be replaced by other promoters (105). To minimize transcriptional interference between the gene’s own promoter and the minigene promoter, we recommend placing the selectable marker gene opposite the target gene’s transcriptional orientation. To minimize potential effects of the selectable marker gene on neighboring genes, and on the phenotype of mice, it is desirable to remove it. This removal can be done by placing Lox P or FRT recognition sequences at both ends of the selectable marker cassette. After transient expression of Cre or Flp-recombinase in ES cells, the marker gene is removed and ES cells become sensitive to the selective agent. This selectivity allows the use of the original targeting vector to create ES cells homozygous at the targeting locus (106,107). If conditional gene targeting is planned, it is advisable to use FRT sites to tag the selectable marker gene (Fig. 4B). For those IF genes that are transcribed in ES cells, very high gene targeting frequencies can be achieved using promoterless selectable marker genes that are placed in the first coding exon, either in frame with the endogenous ATG codon or by positioning the initiation codon of the marker gene downstream from the major transcription start site. If insertion of a promoterless marker gene is required in an exon further 3′ into the gene, an IRES (108) can be incorporated to enable translation of the marker gene. A slight modification of the aforementioned design can be used to introduce a promoterless reporter gene along with the selectable marker to follow the cells carrying the targeted allele. The enhanced green fluorescent protein- or the more sensitive lacZ-reporter genes are convenient but do not necessarily provide quantitative readouts (109,110). There are several ways to introduce subtle alterations, i.e., point mutations, into genes. The mutation can be positioned in one of the homology arms of the targeting vector and be cotransferred along with the selectable marker, provided recombination does not separate the mutation from the selectable marker (Fig. 4B). The selectable marker gene has to be positioned either in a noncoding exon or in an intron, from where it can be removed using Flp or Cre-recombinases, leaving one recombinase recognition site in the genome. In general, cotransfer of mutations work well, provided the distance between the marker
224 Vijayaraj, Söhl, and Magin Fig. 4. (A) Conventional gene-targeting experiment for creation of null allele. Top, genomic locus with two exemplary exons (open boxes). Sequences homologous to replacement-type gene-targeting vector are in black. Middle, targeting vector with short and long arms of homology, flanking a selectable marker gene (gray). The arrow indicates the marker gene promoter and its transcription direction. X indicates regions of crossover. Bottom, targeted locus at which the marker gene has replaced two exons. Arrowheads, primers used for PCR detection of homologous recombinants. Dotted line, predicted PCR product. (B) Introduction of subtle alteration by co-transfer. Top, genomic locus. Middle,
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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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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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