Target Discovery and Validation Reviews and Protocols

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8 Hammerhead Ribozyme-Based Target Discovery Masayuki Sano and Kazunari Taira Summary With the accumulation of vast amounts of data as a result of the sequencing of the human genome, it is necessary to identify human genes that are involved in various cellular, developmental, and disease-related processes and to clarify their functions and potential utility as targets in the treatment of disease. Identification methods based on the use of hammerhead and hairpin ribozymes have received increasing attention as possible tools for the rapid identification of key genes involved in biological processes. This chapter describes the method known as gene-discovery by a hammerhead ribozyme library for elucidation of the gene function. Use of this technology has already revealed new insights into several important biological phenomena. 1. Introduction A hammerhead ribozyme is a small RNA molecule with catalytic activity and it can be engineered to optimize its activity in the intracellular environment. The hammerhead ribozyme recognizes a target gene via interactions between the binding arms of the ribozyme and the gene transcript, and it can cleave any RNA substrate that contains an NUH triple, where N is any nucleotide and H is A, C, or U. To date, numerous studies have been directed towards applications of ribozymes in vivo, and many successful experiments suppressing genes of interest have been reported (1,2). In our laboratory, successful inactivation of genes by hammerhead ribozymes in living cells has been achieved by using an appropriate expression cassette (Fig. 1A). The use of the human tRNAVal promoter ensures a high level of expression of the ribozymes, and an appropriate design of the expression cassette leads to the intracellular stability and cytoplasmic transport of the ribozyme transcripts (3–5). These features would apparently be advantageous for the exploitation of ribozymes in vivo. From: Methods in Molecular Biology, vol. 360, Target Discovery and Validation Reviews and Protocols Volume I, Emerging Strategies for Targets and Biomarker Discovery Edited by: M. Sioud © Humana Press Inc., Totowa, NJ 143
144 Fig. 1. The ribozyme-expression system. (A) The tRNA Val portion is attached to the hammerhead ribozyme (indicated by the bold letters) because the promoters of the gene, namely A and B boxes, are internal. (B) A plasmid for cloning of randomized ribozymes. The promoter of a human gene for tRNA Val is inserted into the pPUR plasmid. The restriction enzyme sites, Csp45I and KpnI, are used for cloning.
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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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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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226 Vijayaraj, Söhl, and Magin gen
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230 Vijayaraj, Söhl, and Magin f.
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234 Vijayaraj, Söhl, and Magin b.
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240 Vijayaraj, Söhl, and Magin alt
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242 Table 3 Time Schedule For Aggre
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250 Vijayaraj, Söhl, and Magin 101
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12 The HUVEC/Matrigel Assay An In V
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256 Skovseth, Küchler, and Haralds
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270 Iversen and Sørensen witnessed
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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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