by Judy, Yuet-wa Chan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 107-111). / Acknowledgements --- p.i / Contents --- p.ii-iv / Abstract --- p.v-vii / Abbreviations --- p.ix / List of figures and tables --- p.x-xii / Chapter Chapter One: --- Introduction --- p.1-20 / Chapter 1.1 --- Facilitative Glucose Transporter Family (GLUT) / Chapter 1.2 --- Sequence and characterization of GLUT / Chapter 1.3 --- Overexpression of GLUT 1 in human cancer cells / Chapter 1.4 --- Inhibition of gene expression by antisense nucleic acid / Chapter 1.5 --- Types of antisense nucleic acids / Chapter 1.5.1 --- Nuclear expression of RNA by engineered antisense genes / Chapter 1.5.2 --- Antisense oligonucleotides / Chapter 1.6 --- Use of antisense oligomers in cell culture system / Chapter 1.7 --- Modification of antisense oligonucleotides / Chapter 1.8 --- Length and sequence selection of antisense oligomers / Chapter 1.9 --- Controls for measuring antisense effect / Chapter 1.10 --- Internalization and targeting of oligonucleotides / Chapter 1.11 --- Possible action mechanisms of antisense nucleotides / Chapter 1.12 --- Clinical applications of antisense approach / Chapter 1.13 --- Aim of the project / Chapter Chapter Two: --- Materials and Methods --- p.21-45 / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Cell line and culture medium / Chapter 2.1.1a --- Cell line / Chapter 2.1.1b --- Culture medium / Chapter 2.1.2 --- Reagents and Buffers / Chapter 2.1.2a --- Phosphate-Buffered Saline (PBS) / Chapter 2.1.2b --- 50XTAE Buffer / Chapter 2.1.2c --- Tris-EDTA Buffer / Chapter 2.1.2d --- MTT solution / Chapter 2.1.2e --- Lipofectin Reagent / Chapter 2.1.3 --- Reagents for Northern Analysis / Chapter 2.1.3a --- DEPC-treated water (0.1% DEPC) / Chapter 2.1.3b --- 20X SSC / Chapter 2.1.3c --- 20X SSPE / Chapter 2.1.3d --- 10X Formaldehyde gel-running buffer / Chapter 2.1.3e --- Formaldehyde gel-loading buffer / Chapter 2.1.3f --- Prehybridization buffer / Chapter 2.1.3g --- Hybridization buffer / Chapter 2.2 --- Methods / Chapter 2.2.1 --- Synthesis of oligonucleotides and phosphorothioated oligonucleotides / Chapter 2.2.2 --- Cloning of human GLUT 1 cDNA into pRc/CMV expression vector at sense and antisense orientation / Chapter 2.2.2a --- Primer designed for cloning of sense and antisense GLUT 1 cDNA / Chapter 2.2.2b --- Isolation of sense and antisense GLUT 1 clone by PCR / Chapter 2.2.2c --- Restriction Digestion / Chapter 2.2.2d --- Purification of Restriction Digested DNA / Chapter 2.2.2e --- DNA Ligation / Chapter 2.2.2f --- Preparation of competent bacterial cells for transformation / Chapter 2.2.2g --- Plasmid DNA Transformation / Chapter 2.2.3 --- Large scale preparation of plasmid DNA / Chapter 2.2.4 --- Formation of Lipofectin-encapsulated oligonucleotides / Chapter 2.2.5 --- [32P]-labeled oligonucleotides uptake assay / Chapter 2.2.6 --- Methods to monitor antisense effect / Chapter 2.2.6a --- MTT assay / Chapter 2.2.6b --- Northern Analysis / Chapter (i) --- Preparation of radiolabeled probe / Chapter (ii) --- Isolation of total RNA from HL-60 cells / Chapter (iii) --- Separation of total RNA by eletrophoresis and blotting onto a membrane / Chapter (iv) --- Prehybridization of the Northern blot / Chapter (v) --- Hybridization of the Northern blot / Chapter 2.2.6c --- [3H]-deoxyglucose uptake assay / Chapter Chapter Three: --- Results --- p.46-88 / Chapter 3.1 --- Synthesis of Oligonucleotides / Chapter 3.2 --- Multiple alignment of cDNA sequence of Glucose Transporter isoforms / Chapter 3.3 --- [32P]-labeled oligonucleotide uptake assay / Chapter 3.4 --- Antisense oligonucleotides designed against different regions of GLUT 1 cDNA sequence / Chapter 3.4.1 --- Effects on HL-60 cell proliferation / Chapter 3.4.2 --- Effects on GLUT 1 mRNA level / Chapter 3.5 --- The effects of different oligonucleotide concentrations on HL- 60cell proliferation / Chapter 3.6 --- The effects of modified oligonucleotides on HL-60 cell proliferation / Chapter 3.7 --- The effects of different oligonucleotide lengths on HL-60 cell proliferation / Chapter 3.8 --- [3H]-deoxyglucose uptake assay / Chapter 3.9 --- Cloning of sense and antisense GLUT 1 cDNA into pRc/CMV vector / Chapter 3.10 --- Inhibition of GLUT 1 gene expression by expressed antisense nucleotides / Chapter Chapter Four: --- Discussion --- p.89-106 / Chapter 4.1 --- Importance of GLUT 1 gene / Chapter 4.2 --- HL-60: the target cancer cell line / Chapter 4.3 --- "Importance of ""Antisense Approach""" / Chapter 4.4 --- Optimization of condition for antisense inhibition by oligonucleotides / Chapter 4.4.1 --- Oligonucleotide length / Chapter 4.4.2 --- Oligonucleotide Modification / Chapter 4.4.3 --- Sequence selection / Chapter 4.4.4 --- Uptake efficiency / Chapter 4.5 --- Intracelluar distribution of oligonucleotides / Chapter 4.6 --- Inhibition of GLUT 1 gene expression by expressed antisense nucleotides / Chapter 4.7 --- Mechanisms for antisense inhibition of gene expression / Chapter 4.8 --- Further Directions / References --- p.107-117
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_321899 |
| Date | January 1997 |
| Contributors | Chan, Judy Yuet-wa., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, viii, 111 leaves : ill. (some mounted) ; 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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