Cheng Tat Cheung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 106-114). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of content --- p.iii / Abbreviations --- p.iv / List of figures --- p.v / List of tables --- p.vi / Chapter Chapter One --- Introduction / Chapter 1.1 --- Ribozyme --- p.1 / Chapter 1.1.1 --- RNA world hypothesis --- p.2 / Chapter 1.1.2 --- Hammerhead ribozyme --- p.3 / Chapter 1.1.3 --- Applications of hammerhead ribozymes --- p.4 / Chapter 1.1.4 --- Allosteric ribozyme --- p.4 / Chapter 1.1.5 --- Ribozyme screening system --- p.5 / Chapter 1.2 --- Other RNA as gene silencing agents --- p.7 / Chapter 1.2.1 --- RNAi --- p.7 / Chapter 1.2.2 --- Antisense RNA --- p.10 / Chapter 1.3 --- Project Overview --- p.11 / Chapter 1.3.1 --- Construction of anti-GFP ribozymes and their in vitro and in vivo studies --- p.11 / Chapter 1.3.2 --- Construction of anti-Elfin ribozyme and its application on gene silencing study --- p.11 / Chapter Chapter Two --- Materials and Methods / Chapter 2.1 --- Cloning techniques --- p.13 / Chapter 2.1.1 --- Polymerase Chain Reaction (PCR) --- p.13 / Chapter 2.1.2 --- Restriction digestion of DNA --- p.13 / Chapter 2.1.3 --- Ligation of DNA fragments --- p.14 / Chapter 2.1.4 --- Preparation of competent cells --- p.15 / Chapter 2.1.5 --- Transformation of competent cells --- p.16 / Chapter 2.1.6 --- Gel extraction --- p.16 / Chapter 2.1.7 --- Plasmid preparation --- p.17 / Chapter 2.1.7.1 --- Mini scale plasmid preparation --- p.17 / Chapter 2.1.7.2 --- Medium scale plasmid preparation --- p.19 / Chapter 2.1.8 --- DNA agarose gel electrophoresis --- p.20 / Chapter 2.1.9 --- Buffer and reagents --- p.21 / Chapter 2.2 --- In vitro cleavage of target RNA by ribozymes --- p.22 / Chapter 2.2.1 --- In vitro transcription of target RNA and ribozymes --- p.22 / Chapter 2.2.2 --- Purification of transcription products --- p.22 / Chapter 2.2.3 --- Ribozymatic cleavage reaction --- p.24 / Chapter 2.2.4 --- Preparation of RNA size marker templates --- p.24 / Chapter 2.2.5 --- Urea-acrylamide gel electrophoresis --- p.25 / Chapter 2.2.6 --- Autoradiography --- p.26 / Chapter 2.2.7 --- Buffer and reagents --- p.26 / Chapter 2.3 --- Detection of cellular RNA expression RT-PCR detection --- p.28 / Chapter 2.3.1 --- RNA extraction --- p.28 / Chapter 2.3.2 --- DNase I digestion --- p.29 / Chapter 2.3.3 --- Reverse transcription --- p.29 / Chapter 2.4 --- Mammalian cell culture techniques --- p.31 / Chapter 2.4.1 --- Transfection into mammalian cells --- p.31 / Chapter 2.4.2 --- Counting the number of cells --- p.32 / Chapter 2.4.2 --- Buffer and reagents --- p.32 / Chapter Chapter Three --- Construction of anti-GFP ribozymes and their in vitro and in vivo studies / Chapter 3.1 --- Introduction --- p.34 / Chapter 3.1.1 --- Objectives --- p.34 / Chapter 3.1.2 --- Why anti-GFP ribozymes? --- p.34 / Chapter 3.2 --- Construction of anti-GFP ribozymes that are active in vitro --- p.36 / Chapter 3.2.1 --- Design of the anti-GFP ribozymes --- p.36 / Chapter 3.2.2 --- Construction of DNA templates for in vitro transcription --- p.40 / Chapter 3.2.3 --- GFP RNA was successfully cleaved by ribozymes --- p.45 / Chapter 3.3 --- In vivo activities of anti-GFP ribozymes --- p.48 / Chapter 3.3.1 --- Design of systems that detected anti-GFP ribozyme activities in --- p.48 / Chapter 3.4 --- The first trial - system α --- p.50 / Chapter 3.4.1 --- Cloning of ribozymes into pACYC184 --- p.51 / Chapter 3.4.2 --- IPTG interfered with GFP expression --- p.54 / Chapter 3.5 --- The second trial - system β --- p.57 / Chapter 3.5.1 --- Insertion of new multiple cloning sites into pET3d --- p.58 / Chapter 3.5.2 --- Cloning of GFP into pET-neu --- p.60 / Chapter 3.5.3 --- GFP expression was interfered by the additional T7 promoter --- p.62 / Chapter 3.6 --- The third trial - system δ --- p.65 / Chapter 3.6.1 --- Insertion of a new cloning site into pET-neu --- p.66 / Chapter 3.6.2 --- Cloning of GFP and ribozymes into pET-fn --- p.68 / Chapter 3.6.3 --- Ribozymes sequence upstream of GFP interfered with GFP expression / Chapter 3.7 --- The fourth trial - system co --- p.73 / Chapter 3.7.1 --- Insertion of new cloning sites into pET-neu --- p.74 / Chapter 3.7.2 --- Cloning of GFP and ribozymes into pET-nr --- p.76 / Chapter 3.7.3 --- Anti-GFP ribozymes did not turn off green fluorescence of GFP --- p.79 / Chapter 3.7.4 --- No in vivo cleavage of GFP was detected --- p.81 / Chapter 3.8 --- Summary --- p.83 / Chapter Chapter Four --- Construction of an anti-Elfin ribozyme and its application on gene silencing study / Chapter 4.1 --- Introduction --- p.84 / Chapter 4.1.1 --- Objectives --- p.84 / Chapter 4.1.2 --- Elfin --- p.84 / Chapter 4.1.3 --- Experimental plan --- p.85 / Chapter 4.2 --- In vitro cleavage of Elfin RNA by ribozyme --- p.86 / Chapter 4.2.1 --- Design of anti-Elfin ribozyme --- p.86 / Chapter 4.2.2 --- Preparation of DNA template for in vitro transcription --- p.88 / Chapter 4.2.3 --- Successful in vitro cleavage of Elfin RNA by ribozyme --- p.88 / Chapter 4.3 --- In vivo gene silencing studies of RNA tools --- p.90 / Chapter 4.3.1 --- Design of antisense RNA --- p.90 / Chapter 4.3.2 --- Design of siRNA --- p.92 / Chapter 4.3.3 --- Cloning of RNA tools into pSilencer --- p.94 / Chapter 4.3.4 --- Cloning of a neomycin resistance gene into pSilencer-R --- p.94 / Chapter 4.3.5 --- Elfin RNA was not down-regulated --- p.97 / Chapter 4.4 --- Summary --- p.100 / Chapter Chapter 5 --- Discussions --- p.101 / Reference / Appendix
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_324368 |
| Date | January 2003 |
| Contributors | Cheng, Tat Cheung., Chinese University of Hong Kong Graduate School. Division of Molecular Biotechnology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, 118 leaves : ill. (some col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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