Lee Yiu Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 178-183). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Abstract --- p.iii / Abstract (Chinese version) --- p.vi / Table of contents --- p.viii / List of Abbreviations --- p.xvi / List of Figures --- p.xviii / List of Tables --- p.xxiv / Chapter Chapter 1 --- INTRODUCTON / Chapter 1.1 --- Cytochrome P450 (P450) --- p.1 / Chapter 1.1.1 --- Cytochrome P450 family --- p.1 / Chapter 1.1.2 --- Role in metabolism --- p.4 / Chapter 1.1.3 --- P450 catalytic cycle --- p.6 / Chapter 1.2 --- NADPH-cytochrome P450 reductase (RED) --- p.6 / Chapter 1.2.1 --- Characterization and distribution --- p.6 / Chapter 1.2.2 --- Structural and functional domains --- p.8 / Chapter 1.2.3 --- Role in P450 catalytic cycle --- p.10 / Chapter 1.3 --- Drug metabolism --- p.10 / Chapter 1.3.1 --- Understanding of drug metabolism is important for drug development --- p.10 / Chapter 1.3.2 --- Role of P450 in drug metabolism --- p.12 / Chapter 1.4 --- Production of RED knockout in vivo mouse model for screening of P450-dependent new drugs --- p.13 / Chapter 1.4.1 --- Background --- p.13 / Chapter 1.4.2 --- Gene targeting --- p.13 / Chapter 1.4.3 --- Gene targeting vector --- p.15 / Chapter 1.4.3.1 --- Classical knockout --- p.15 / Chapter 1.4.3.2 --- Conditional knockout --- p.19 / Chapter 1.4.4 --- Gene knockout mice and its use --- p.21 / Chapter Chapter 2 --- OBJECTIVES --- p.22 / Chapter Chapter 3 --- MATERIALS AND METHODS --- p.24 / Chapter 3.1 --- Preparation of RED cDNA by RT-PCR --- p.24 / Chapter 3.1.1 --- Total RNA isolation --- p.24 / Chapter 3.1.1.1 --- Materials --- p.24 / Chapter 3.1.1.2 --- Methods --- p.24 / Chapter 3.1.2 --- Reverse transcription- polymerase chain reaction (RT-PCR) --- p.25 / Chapter 3.1.2.1 --- Materials --- p.25 / Chapter 3.1.2.2 --- Methods --- p.25 / Chapter 3.1.3 --- T/A cloning of RED cDNA --- p.28 / Chapter 3.1.3.1 --- Materials --- p.28 / Chapter 3.1.3.2 --- Methods --- p.28 / Chapter 3.1.4 --- Midi-preparation of RED cDNA clone --- p.32 / Chapter 3.1.4.1 --- Materials --- p.33 / Chapter 3.1.4.2 --- Methods --- p.33 / Chapter 3.1.5 --- Confirmation of RED cDNA clone --- p.34 / Chapter 3.1.5.1 --- Restriction enzyme mapping --- p.34 / Chapter 3.1.5.1.1 --- Materials --- p.34 / Chapter 3.1.5.1.2 --- Methods --- p.34 / Chapter 3.1.5.2 --- DNA sequencing of RED cDNA sequence --- p.35 / Chapter 3.1.5.2.1 --- Materials --- p.35 / Chapter 3.1.5.2.2 --- Methods --- p.35 / Chapter 3.1.6 --- Preparation and purification of RED cDNA for probe labeling --- p.38 / Chapter 3.1.6.1 --- Materials --- p.38 / Chapter 3.1.6.2 --- Methods --- p.38 / Chapter 3.1.7 --- Non-radioactive random-primed labeling of RED cDNA --- p.39 / Chapter 3.1.7.1 --- Materials --- p.39 / Chapter 3.1.7.2 --- Methods --- p.39 / Chapter 3.2 --- Isolation of RED gene by genomic library screening --- p.40 / Chapter 3.2.1 --- Titering of genomic library --- p.41 / Chapter 3.2.1.1 --- Materials --- p.41 / Chapter 3.2.1.2 --- Methods --- p.41 / Chapter 3.2.2 --- Primary screening of genomic library by RED cDNA probe --- p.42 / Chapter 3.2.2.1 --- Plaque lift --- p.42 / Chapter 3.2.2.1.1 --- Materials --- p.42 / Chapter 3.2.2.1.2 --- Methods --- p.42 / Chapter 3.2.2.2 --- Proteinase K treatment --- p.43 / Chapter 3.2.2.2.1 --- Materials --- p.43 / Chapter 3.2.2.2.2 --- Methods --- p.43 / Chapter 3.2.2.3 --- "Pre-hybridization, hybridization and detection" --- p.44 / Chapter 3.2.2.3.1 --- Materials --- p.44 / Chapter 3.2.2.3.2 --- Methods --- p.44 / Chapter 3.3 --- Isolation of RED by hybridization screening by Genome System Inc. --- p.45 / Chapter 3.4 --- Characterization of BAC clones containing RED genomic DNA fragments commercially obtained from Genome System Inc. --- p.45 / Chapter 3.4.1 --- Large scale preparation of BAC DNA --- p.45 / Chapter 3.4.1.1 --- Materials --- p.47 / Chapter 3.4.1.2 --- Methods --- p.47 / Chapter 3.4.2 --- Restriction enzyme mappings and Southern blotting analysis of BAC DNA fragments --- p.47 / Chapter 3.4.2.1 --- Materials --- p.48 / Chapter 3.4.2.2 --- Methods --- p.48 / Chapter 3.4.3 --- Shot-gun sub-cloning of RED genomic DNA fragments from BAC clone in pGEM®-3Z vector --- p.49 / Chapter 3.4.3.1 --- Preparation of cloning vector and DNA insert for ligation --- p.50 / Chapter 3.4.3.1.1 --- Materials --- p.50 / Chapter 3.4.3.1.2 --- Methods --- p.50 / Chapter 3.4.3.1.2.1 --- Cloning vectors --- p.50 / Chapter 3.4.3.1.2.2 --- DNA inserts --- p.52 / Chapter 3.4.3.2 --- Preparation of competent cells and transformation --- p.52 / Chapter 3.4.3.2.1 --- Materials --- p.52 / Chapter 3.4.3.2.2 --- Methods --- p.53 / Chapter 3.4.3.3 --- Screening for positive recombinant clones --- p.54 / Chapter 3.4.3.3.1 --- Picking of colonies randomly from the agar plates (method 1) --- p.54 / Chapter 3.4.3.3.1.1 --- Materials --- p.54 / Chapter 3.4.3.3.1.2 --- Methods --- p.54 / Chapter 3.4.3.3.2 --- Colony lifts and hybridization with RED cDNA probes (method 2) --- p.55 / Chapter 3.4.3.3.2.1 --- Materials --- p.55 / Chapter 3.4.3.3.2.2 --- Methods --- p.55 / Chapter 3.5 --- Restriction enzyme mappings and Southern blotting analysis of RED gene subcloned in pGEM®-3Z vector --- p.56 / Chapter 3.5.1 --- Materials --- p.56 / Chapter 3.5.2 --- Methods --- p.56 / Chapter 3.6 --- Exon mappings of the RED genomic DNA fragments by PCR --- p.57 / Chapter 3.6.1 --- Materials --- p.57 / Chapter 3.6.2 --- Methods --- p.57 / Chapter 3.7 --- Construction of gene targeting vector --- p.57 / Chapter 3.7.1 --- Gene targeting vectors la and lb derived from clone H (strategy 1) --- p.60 / Chapter 3.7.1.1 --- Sub-cloning 3.65 kb Hind Ill/Hind III RED gene fragment to pGEM®-3Z vector --- p.60 / Chapter 3.7.1.1.1 --- Materials --- p.62 / Chapter 3.7.1.1.2 --- Methods --- p.62 / Chapter 3.7.1.2 --- Deletion of exonic sequence of RED gene and modification of the digested restriction end to Xho I site --- p.62 / Chapter 3.7.1.2.1 --- Materials --- p.63 / Chapter 3.7.1.2.2 --- Methods --- p.63 / Chapter 3.7.1.3 --- Preparation of neo cassette --- p.63 / Chapter 3.7.1.3.1 --- Materials --- p.64 / Chapter 3.7.1.3.2 --- Methods --- p.64 / Chapter 3.7.1.4 --- Cloning of neo cassette --- p.66 / Chapter 3.7.1.4.1 --- Methods --- p.66 / Chapter 3.7.1.5 --- Sub-cloning the neo cassette containing RED genomic fragment to pMCI-Thymidine kinase (TK) Poly A vector --- p.67 / Chapter 3.7.1.5.1 --- Materials --- p.67 / Chapter 3.7.1.5.2 --- Methods --- p.67 / Chapter 3.7.2 --- "Gene targeting vectors 2a/2b, 3a/3b and 4a derived from clone X8 (strategy 2,3 and 4 respectively)" --- p.67 / Chapter 3.8 --- Preparation and testing the genomic probes for screening recombinant embryonic stem (ES) cells --- p.73 / Chapter 3.8.1 --- Cloning of genomic probes --- p.73 / Chapter 3.8.1.1 --- Materials --- p.73 / Chapter 3.8.1.2 --- Methods --- p.73 / Chapter 3.8.2 --- Purification of DNA for labeling --- p.78 / Chapter 3.8.2.1 --- Materials --- p.78 / Chapter 3.8.2.2 --- Methods --- p.78 / Chapter 3.8.3 --- ECF random prime labeling of genomic probes --- p.79 / Chapter 3.8.3.1 --- Materials --- p.79 / Chapter 3.8.3.2 --- Methods --- p.79 / Chapter 3.8.4 --- Restriction enzyme digestion of genomic DNA and Southern blotting --- p.80 / Chapter 3.8.4.1 --- Materials --- p.80 / Chapter 3.8.4.2 --- Methods --- p.80 / Chapter 3.8.5 --- Testing the specificity of genomic probes --- p.80 / Chapter 3.8.5.1 --- Materials --- p.80 / Chapter 3.8.5.2 --- Methods --- p.80 / Chapter Chapter 4 --- RESULTS --- p.86 / Chapter 4.1 --- Total RNA isolation and RT-PCR of RED cDNAs --- p.86 / Chapter 4.2 --- Confirmation of the RT-PCR RED cDNA clone --- p.86 / Chapter 4.2.1 --- Restriction enzyme mapping --- p.86 / Chapter 4.2.2 --- DNA sequencing --- p.86 / Chapter 4.3 --- Genomic library screening of RED gene --- p.90 / Chapter 4 4 --- Restriction enzyme mappings and Southern blotting analysis of RED Gene containing BAC clone from Genome System Inc. --- p.90 / Chapter 4.5 --- Shot-gun sub-cloning of RED gene containing genomic DNA fragments to pGEM®-3Z vectors --- p.93 / Chapter 4.5.1 --- Cloning of Hind III cut RED gene fragment --- p.93 / Chapter 4.5.2 --- Cloning of Xba I cut RED gene fragment --- p.93 / Chapter 4.5.3 --- Cloning of EcoR I cut RED gene fragment --- p.95 / Chapter 4.6 --- Identification of RED exons in the shot-gun sub-cloning clones by PCR --- p.95 / Chapter 4.7 --- Construction of restriction enzyme maps of the RED gene containing clones --- p.100 / Chapter 4.7.1 --- Clone H --- p.100 / Chapter 4.7.1.1 --- Single restriction enzyme digestions and Southern blotting --- p.100 / Chapter 4.7.1.2 --- Double restriction enzyme digestions and Southern blotting --- p.100 / Chapter 4.7.1.3 --- Restriction enzyme map --- p.101 / Chapter 4.7.2 --- Clone X8 --- p.101 / Chapter 4.7.2.1 --- Single restriction enzyme digestions and Southern blotting --- p.101 / Chapter 4.7.2.2 --- Double restriction enzyme digestion and Southern blotting --- p.104 / Chapter 4.7.2.3 --- Restriction enzyme map --- p.104 / Chapter 4.7.3 --- Clone El4 --- p.105 / Chapter 4.7.3.1 --- Single restriction enzyme digestions and Southern blotting --- p.105 / Chapter 4.7.3.2 --- Double restriction enzyme digestion and Southern blotting --- p.108 / Chapter 4.7.3.3 --- Restriction enzyme map --- p.108 / Chapter 4.8 --- Construction of gene targeting vector --- p.108 / Chapter 4.8.1 --- Gene targeting vector based on the clone H (strategy 1) with deletion of RED exon 16 --- p.113 / Chapter 4.8.1.1 --- Cloning a smaller RED genomic DNA into pGEM®-3Z vectors --- p.113 / Chapter 4.8.1.2 --- Replacement of exon of RED gene by neo cassette --- p.113 / Chapter 4.8.1.3 --- Cloning to TK vector --- p.113 / Chapter 4.8.2 --- Targeting vector based on the clone X8 --- p.124 / Chapter 4.8.2.1 --- Strategy 2 (deletion of RED exon 4) --- p.124 / Chapter 4.8.2.1.1 --- Cloning 3.9 kb Kpn I/Hinc II RED genomic DNA into pGEM®-3Z vectors --- p.124 / Chapter 4.8.2.1.2 --- Replacement of exon of RED gene by neo cassette --- p.124 / Chapter 4.8.2.1.3 --- Cloning to TK vector --- p.124 / Chapter 4.8.2.2 --- Strategy 3 (deletion of RED exon 5-8) --- p.136 / Chapter 4.8.2.2.1 --- Cloning the genomic DNA into pGEM®-3Z vectors --- p.136 / Chapter 4.8.2.2.2 --- Replacement of exon of RED gene by neo cassette --- p.136 / Chapter 4.8.2.2.3 --- Cloning to TK vector --- p.136 / Chapter 4.8.2.3 --- Strategy 4 (deletion of RED exon 7-10) --- p.136 / Chapter 4.8.2.3.1 --- Cloning the genomic DNA into pGEM®-3Z vectors --- p.136 / Chapter 4.8.2.3.2 --- Replacement of exon of RED gene by neo cassette --- p.152 / Chapter 4.8.2.3.3 --- Cloning to TK vector --- p.152 / Chapter 4.9 --- Testing for the specificity of genomic DNA probes --- p.152 / Chapter 4.9.1 --- Preparation of restriction enzyme digested genomic DNA --- p.152 / Chapter 4.9.2 --- Hybridization of the probes to genomic DNA --- p.163 / Chapter Chapter 5 --- DISCUSSION --- p.167 / Chapter 5.1 --- Proposed significant of RED knockout mice for new drug screening --- p.167 / Chapter 5.2 --- Experimental problems --- p.168 / Chapter 5.2.1 --- Genomic library screening --- p.168 / Chapter 5.2.2 --- Cloning --- p.168 / Chapter 5.3 --- RED gene targeting vector construction / Chapter 5.3.1 --- Isolation of RED gene for gene targeting vectors construction --- p.169 / Chapter 5.3.2 --- Deletion of different exons in different RED gene targeting vectors --- p.169 / Chapter 5.3.3 --- Components in the targeting vectors --- p.170 / Chapter 5.3.4 --- Enhancements of homologous recombination --- p.171 / Chapter Chapter 6 --- CONCLUSIONS --- p.173 / Chapter Chapter 7 --- FUTURE STUDIES --- p.175 / Chapter 7.1 --- Identification of the sizes of RED gene introns --- p.175 / Chapter 7.2 --- Production of RED knockout mice --- p.175 / Chapter 7.3 --- Characterization of RED knockout mice --- p.175 / Chapter 7.4 --- Conditional gene knockout for RED gene --- p.177 / REFERENCES --- p.178 / APPENDIX --- p.184
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_323529 |
| Date | January 2001 |
| Contributors | Lee, Yiu Fai., Chinese University of Hong Kong Graduate School. Division of Biochemistry. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xxiv, 231 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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