Construction of gene targeting vectors for production of Nadph-Cytochrome P450 reductase (red) knockout mice.

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.iiAbstract --- p.iiiAbstract (Chinese version) --- p.viTable of contents --- p.viiiList of Abbreviations --- p.xviList of Figures --- p.xviiiList of Tables --- p.xxivChapter Chapter 1 --- INTRODUCTONChapter 1.1 --- Cytochrome P450 (P450) --- p.1Chapter 1.1.1 --- Cytochrome P450 family --- p.1Chapter 1.1.2 --- Role in metabolism --- p.4Chapter 1.1.3 --- P450 catalytic cycle --- p.6Chapter 1.2 --- NADPH-cytochrome P450 reductase (RED) --- p.6Chapter 1.2.1 --- Characterization and distribution --- p.6Chapter 1.2.2 --- Structural and functional domains --- p.8Chapter 1.2.3 --- Role in P450 catalytic cycle --- p.10Chapter 1.3 --- Drug metabolism --- p.10Chapter 1.3.1 --- Understanding of drug metabolism is important for drug development --- p.10Chapter 1.3.2 --- Role of P450 in drug metabolism --- p.12Chapter 1.4 --- Production of RED knockout in vivo mouse model for screening of P450-dependent new drugs --- p.13Chapter 1.4.1 --- Background --- p.13Chapter 1.4.2 --- Gene targeting --- p.13Chapter 1.4.3 --- Gene targeting vector --- p.15Chapter 1.4.3.1 --- Classical knockout --- p.15Chapter 1.4.3.2 --- Conditional knockout --- p.19Chapter 1.4.4 --- Gene knockout mice and its use --- p.21Chapter Chapter 2 --- OBJECTIVES --- p.22Chapter Chapter 3 --- MATERIALS AND METHODS --- p.24Chapter 3.1 --- Preparation of RED cDNA by RT-PCR --- p.24Chapter 3.1.1 --- Total RNA isolation --- p.24Chapter 3.1.1.1 --- Materials --- p.24Chapter 3.1.1.2 --- Methods --- p.24Chapter 3.1.2 --- Reverse transcription- polymerase chain reaction (RT-PCR) --- p.25Chapter 3.1.2.1 --- Materials --- p.25Chapter 3.1.2.2 --- Methods --- p.25Chapter 3.1.3 --- T/A cloning of RED cDNA --- p.28Chapter 3.1.3.1 --- Materials --- p.28Chapter 3.1.3.2 --- Methods --- p.28Chapter 3.1.4 --- Midi-preparation of RED cDNA clone --- p.32Chapter 3.1.4.1 --- Materials --- p.33Chapter 3.1.4.2 --- Methods --- p.33Chapter 3.1.5 --- Confirmation of RED cDNA clone --- p.34Chapter 3.1.5.1 --- Restriction enzyme mapping --- p.34Chapter 3.1.5.1.1 --- Materials --- p.34Chapter 3.1.5.1.2 --- Methods --- p.34Chapter 3.1.5.2 --- DNA sequencing of RED cDNA sequence --- p.35Chapter 3.1.5.2.1 --- Materials --- p.35Chapter 3.1.5.2.2 --- Methods --- p.35Chapter 3.1.6 --- Preparation and purification of RED cDNA for probe labeling --- p.38Chapter 3.1.6.1 --- Materials --- p.38Chapter 3.1.6.2 --- Methods --- p.38Chapter 3.1.7 --- Non-radioactive random-primed labeling of RED cDNA --- p.39Chapter 3.1.7.1 --- Materials --- p.39Chapter 3.1.7.2 --- Methods --- p.39Chapter 3.2 --- Isolation of RED gene by genomic library screening --- p.40Chapter 3.2.1 --- Titering of genomic library --- p.41Chapter 3.2.1.1 --- Materials --- p.41Chapter 3.2.1.2 --- Methods --- p.41Chapter 3.2.2 --- Primary screening of genomic library by RED cDNA probe --- p.42Chapter 3.2.2.1 --- Plaque lift --- p.42Chapter 3.2.2.1.1 --- Materials --- p.42Chapter 3.2.2.1.2 --- Methods --- p.42Chapter 3.2.2.2 --- Proteinase K treatment --- p.43Chapter 3.2.2.2.1 --- Materials --- p.43Chapter 3.2.2.2.2 --- Methods --- p.43Chapter 3.2.2.3 --- "Pre-hybridization, hybridization and detection" --- p.44Chapter 3.2.2.3.1 --- Materials --- p.44Chapter 3.2.2.3.2 --- Methods --- p.44Chapter 3.3 --- Isolation of RED by hybridization screening by Genome System Inc. --- p.45Chapter 3.4 --- Characterization of BAC clones containing RED genomic DNA fragments commercially obtained from Genome System Inc. --- p.45Chapter 3.4.1 --- Large scale preparation of BAC DNA --- p.45Chapter 3.4.1.1 --- Materials --- p.47Chapter 3.4.1.2 --- Methods --- p.47Chapter 3.4.2 --- Restriction enzyme mappings and Southern blotting analysis of BAC DNA fragments --- p.47Chapter 3.4.2.1 --- Materials --- p.48Chapter 3.4.2.2 --- Methods --- p.48Chapter 3.4.3 --- Shot-gun sub-cloning of RED genomic DNA fragments from BAC clone in pGEM®-3Z vector --- p.49Chapter 3.4.3.1 --- Preparation of cloning vector and DNA insert for ligation --- p.50Chapter 3.4.3.1.1 --- Materials --- p.50Chapter 3.4.3.1.2 --- Methods --- p.50Chapter 3.4.3.1.2.1 --- Cloning vectors --- p.50Chapter 3.4.3.1.2.2 --- DNA inserts --- p.52Chapter 3.4.3.2 --- Preparation of competent cells and transformation --- p.52Chapter 3.4.3.2.1 --- Materials --- p.52Chapter 3.4.3.2.2 --- Methods --- p.53Chapter 3.4.3.3 --- Screening for positive recombinant clones --- p.54Chapter 3.4.3.3.1 --- Picking of colonies randomly from the agar plates (method 1) --- p.54Chapter 3.4.3.3.1.1 --- Materials --- p.54Chapter 3.4.3.3.1.2 --- Methods --- p.54Chapter 3.4.3.3.2 --- Colony lifts and hybridization with RED cDNA probes (method 2) --- p.55Chapter 3.4.3.3.2.1 --- Materials --- p.55Chapter 3.4.3.3.2.2 --- Methods --- p.55Chapter 3.5 --- Restriction enzyme mappings and Southern blotting analysis of RED gene subcloned in pGEM®-3Z vector --- p.56Chapter 3.5.1 --- Materials --- p.56Chapter 3.5.2 --- Methods --- p.56Chapter 3.6 --- Exon mappings of the RED genomic DNA fragments by PCR --- p.57Chapter 3.6.1 --- Materials --- p.57Chapter 3.6.2 --- Methods --- p.57Chapter 3.7 --- Construction of gene targeting vector --- p.57Chapter 3.7.1 --- Gene targeting vectors la and lb derived from clone H (strategy 1) --- p.60Chapter 3.7.1.1 --- Sub-cloning 3.65 kb Hind Ill/Hind III RED gene fragment to pGEM®-3Z vector --- p.60Chapter 3.7.1.1.1 --- Materials --- p.62Chapter 3.7.1.1.2 --- Methods --- p.62Chapter 3.7.1.2 --- Deletion of exonic sequence of RED gene and modification of the digested restriction end to Xho I site --- p.62Chapter 3.7.1.2.1 --- Materials --- p.63Chapter 3.7.1.2.2 --- Methods --- p.63Chapter 3.7.1.3 --- Preparation of neo cassette --- p.63Chapter 3.7.1.3.1 --- Materials --- p.64Chapter 3.7.1.3.2 --- Methods --- p.64Chapter 3.7.1.4 --- Cloning of neo cassette --- p.66Chapter 3.7.1.4.1 --- Methods --- p.66Chapter 3.7.1.5 --- Sub-cloning the neo cassette containing RED genomic fragment to pMCI-Thymidine kinase (TK) Poly A vector --- p.67Chapter 3.7.1.5.1 --- Materials --- p.67Chapter 3.7.1.5.2 --- Methods --- p.67Chapter 3.7.2 --- "Gene targeting vectors 2a/2b, 3a/3b and 4a derived from clone X8 (strategy 2,3 and 4 respectively)" --- p.67Chapter 3.8 --- Preparation and testing the genomic probes for screening recombinant embryonic stem (ES) cells --- p.73Chapter 3.8.1 --- Cloning of genomic probes --- p.73Chapter 3.8.1.1 --- Materials --- p.73Chapter 3.8.1.2 --- Methods --- p.73Chapter 3.8.2 --- Purification of DNA for labeling --- p.78Chapter 3.8.2.1 --- Materials --- p.78Chapter 3.8.2.2 --- Methods --- p.78Chapter 3.8.3 --- ECF random prime labeling of genomic probes --- p.79Chapter 3.8.3.1 --- Materials --- p.79Chapter 3.8.3.2 --- Methods --- p.79Chapter 3.8.4 --- Restriction enzyme digestion of genomic DNA and Southern blotting --- p.80Chapter 3.8.4.1 --- Materials --- p.80Chapter 3.8.4.2 --- Methods --- p.80Chapter 3.8.5 --- Testing the specificity of genomic probes --- p.80Chapter 3.8.5.1 --- Materials --- p.80Chapter 3.8.5.2 --- Methods --- p.80Chapter Chapter 4 --- RESULTS --- p.86Chapter 4.1 --- Total RNA isolation and RT-PCR of RED cDNAs --- p.86Chapter 4.2 --- Confirmation of the RT-PCR RED cDNA clone --- p.86Chapter 4.2.1 --- Restriction enzyme mapping --- p.86Chapter 4.2.2 --- DNA sequencing --- p.86Chapter 4.3 --- Genomic library screening of RED gene --- p.90Chapter 4 4 --- Restriction enzyme mappings and Southern blotting analysis of RED Gene containing BAC clone from Genome System Inc. --- p.90Chapter 4.5 --- Shot-gun sub-cloning of RED gene containing genomic DNA fragments to pGEM®-3Z vectors --- p.93Chapter 4.5.1 --- Cloning of Hind III cut RED gene fragment --- p.93Chapter 4.5.2 --- Cloning of Xba I cut RED gene fragment --- p.93Chapter 4.5.3 --- Cloning of EcoR I cut RED gene fragment --- p.95Chapter 4.6 --- Identification of RED exons in the shot-gun sub-cloning clones by PCR --- p.95Chapter 4.7 --- Construction of restriction enzyme maps of the RED gene containing clones --- p.100Chapter 4.7.1 --- Clone H --- p.100Chapter 4.7.1.1 --- Single restriction enzyme digestions and Southern blotting --- p.100Chapter 4.7.1.2 --- Double restriction enzyme digestions and Southern blotting --- p.100Chapter 4.7.1.3 --- Restriction enzyme map --- p.101Chapter 4.7.2 --- Clone X8 --- p.101Chapter 4.7.2.1 --- Single restriction enzyme digestions and Southern blotting --- p.101Chapter 4.7.2.2 --- Double restriction enzyme digestion and Southern blotting --- p.104Chapter 4.7.2.3 --- Restriction enzyme map --- p.104Chapter 4.7.3 --- Clone El4 --- p.105Chapter 4.7.3.1 --- Single restriction enzyme digestions and Southern blotting --- p.105Chapter 4.7.3.2 --- Double restriction enzyme digestion and Southern blotting --- p.108Chapter 4.7.3.3 --- Restriction enzyme map --- p.108Chapter 4.8 --- Construction of gene targeting vector --- p.108Chapter 4.8.1 --- Gene targeting vector based on the clone H (strategy 1) with deletion of RED exon 16 --- p.113Chapter 4.8.1.1 --- Cloning a smaller RED genomic DNA into pGEM®-3Z vectors --- p.113Chapter 4.8.1.2 --- Replacement of exon of RED gene by neo cassette --- p.113Chapter 4.8.1.3 --- Cloning to TK vector --- p.113Chapter 4.8.2 --- Targeting vector based on the clone X8 --- p.124Chapter 4.8.2.1 --- Strategy 2 (deletion of RED exon 4) --- p.124Chapter 4.8.2.1.1 --- Cloning 3.9 kb Kpn I/Hinc II RED genomic DNA into pGEM®-3Z vectors --- p.124Chapter 4.8.2.1.2 --- Replacement of exon of RED gene by neo cassette --- p.124Chapter 4.8.2.1.3 --- Cloning to TK vector --- p.124Chapter 4.8.2.2 --- Strategy 3 (deletion of RED exon 5-8) --- p.136Chapter 4.8.2.2.1 --- Cloning the genomic DNA into pGEM®-3Z vectors --- p.136Chapter 4.8.2.2.2 --- Replacement of exon of RED gene by neo cassette --- p.136Chapter 4.8.2.2.3 --- Cloning to TK vector --- p.136Chapter 4.8.2.3 --- Strategy 4 (deletion of RED exon 7-10) --- p.136Chapter 4.8.2.3.1 --- Cloning the genomic DNA into pGEM®-3Z vectors --- p.136Chapter 4.8.2.3.2 --- Replacement of exon of RED gene by neo cassette --- p.152Chapter 4.8.2.3.3 --- Cloning to TK vector --- p.152Chapter 4.9 --- Testing for the specificity of genomic DNA probes --- p.152Chapter 4.9.1 --- Preparation of restriction enzyme digested genomic DNA --- p.152Chapter 4.9.2 --- Hybridization of the probes to genomic DNA --- p.163Chapter Chapter 5 --- DISCUSSION --- p.167Chapter 5.1 --- Proposed significant of RED knockout mice for new drug screening --- p.167Chapter 5.2 --- Experimental problems --- p.168Chapter 5.2.1 --- Genomic library screening --- p.168Chapter 5.2.2 --- Cloning --- p.168Chapter 5.3 --- RED gene targeting vector constructionChapter 5.3.1 --- Isolation of RED gene for gene targeting vectors construction --- p.169Chapter 5.3.2 --- Deletion of different exons in different RED gene targeting vectors --- p.169Chapter 5.3.3 --- Components in the targeting vectors --- p.170Chapter 5.3.4 --- Enhancements of homologous recombination --- p.171Chapter Chapter 6 --- CONCLUSIONS --- p.173Chapter Chapter 7 --- FUTURE STUDIES --- p.175Chapter 7.1 --- Identification of the sizes of RED gene introns --- p.175Chapter 7.2 --- Production of RED knockout mice --- p.175Chapter 7.3 --- Characterization of RED knockout mice --- p.175Chapter 7.4 --- Conditional gene knockout for RED gene --- p.177REFERENCES --- p.178APPENDIX --- p.18