Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 261-288).

Thesis (M.Sc.). / Written for the Dept. of Human Genetics. Title from title page of PDF (viewed 2008/07/30). Includes bibliographical references.

Thesis (Ph.D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Pharmacology & Toxicology. Title from title-page of electronic thesis. Bibliography: leaves 187-206.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p. 97-121.

Thesis (M.S.)--University of Southern California, 1999. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (M.S.)--University of Southern California, 1999. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

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

The CYP27B1 enzyme that synthesizes 1alpha,25-(OH) 2D, is expressed in chondrocytes, suggesting that local production of 1alpha,25-(OH)2D could play an autocrine or paracrine role in the differentiation of these cells. To test this hypothesis, we have engineered mutant mice that do not express the Cyp27b1 gene in chondrocytes. This led to increased width of the hypertrophic zone of the growth plate at E15.5, increased bone mass in neonatal long bones, and increased expression of the chondrocytic differentiation markers Indian Hedgehog and PTH/PTHrP receptor. VEGF mRNA levels were decreased, accompanied by decreased PECAM-1 immunostaining, suggesting a delay in vascularization. We have also engineered mice overexpressing a Cyp27b1 transgene in chondrocytes. The transgenic mice showed a partial mirror image phenotype compared to the tissue-specific inactivation model. These results support an autocrine/paracrine role of 1alpha,25-(OH) 2D in endochondral ossification and chondrocyte development in vivo. / The CYP24A1 enzyme is involved in the catabolic breakdown of 1alpha,25-(OH)2D but also synthesizes the 24R,25-(OH) 2D metabolite. Studies in chicken suggest a role for 24R,25-(OH) 2D in fracture repair. We induced stabilized transverse mid-diaphysial fractures of the tibia in four-month-old wild-type and Cyp24a1-deficient mice. Examination of the callus sections showed delayed hard callus formation in the homozygous mutant animals compared to wild-type littermates. RT-qPCR showed perturbed levels of type X collagen transcripts in mutant mice at 14 and 21 days post-fracture, reflecting the delayed healing. Rescue of the impaired healing by subcutaneous injection of 24R,25-(OH)2D3 normalized the histological appearance of the callus, static histomorphometric index and type X collagen mRNA expression, while 1alpha,25-(OH)2D 3 did not. These results show that Cyp24a1 deficiency delays fracture repair and strongly suggest that vitamin D metabolites hydroxylated at position 24, such as 24R,25-(OH)2D, play an important role in the mechanisms leading to normal fracture healing.

The proprotein convertase subtilisin/kexin type 9, PCSK9, belongs to the proprotein convertase (PC) family. Human mutations in the gene encoding PCSK9 lead to either familial hyper- or hypocholesterolemia, resulting from a gain or loss of function, respectively. Mice lacking PCSK9 are viable and show a 42% decrease in plasma cholesterol levels. The enzyme triggers the degradation of the low density lipoprotein receptor (LDLR) through a partially unknown mechanism. / PCSK9 is very abundant in the liver and intestine during development and adulthood. Hepatocytes have a capacity to reproduce themselves and, upon injury, can repopulate the liver. For a better understanding of the role of PCSK9 in the liver, partial hepatectomy was performed on Pcsk9 +/+, Pcsk9+/- and Pcsk9-/- mice. The absence of PCSK9 resulted in defective liver regeneration, while wild type (WT) and heterozygous mice had no phenotype. Regeneration defects could be prevented by a high cholesterol diet. PCSK9 deficiency, by contributing to maintaining low circulating cholesterol levels may thus hamper liver regeneration. This knowledge is critical for the analysis of future PCSK9 inhibitors expected to be developed in the near future. / Key words. Proprotein convertase subtilisin/kexin 9 (PCSK9), a familial hyper- or hypocholesterolemia, low density lipoprotein receptor, knockout mouse model, partial hepatectomy.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed Feb. 13, 2009). Includes bibliographical references.