Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2003. / "16th September 2002." Accompanying CD contains 2 MPEG clips with accompanying text, and a copy in PDF format of: Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer / M. Limberis ... [et al.], published in Human gene therapy vol. 13 (2002). Bibliography: leaves xxix-li.

No description available.

The purpose of this study was two-fold. The first objective was to determine if Saccharomyces cerevisiae is a useful system for investigating the expression of T-DNA (it takes several months to obtain sufficient bacteria-free transformed plant tissue to investigate T-DNA transcription). A short fragment of T-DNA carrying T-DNA gene 7 was cloned into a yeast plasmid in an attempt to investigate the expression of gene 7 in yeast. The second objective was to determine the significance of a heat shock related sequence identified in the 5¹ region of T-DNA gene 7. Primer extension analysis, SI nuclease mapping, and Northern hybridizations indicate that transcription of T-DNA gene 7 in yeast is different from that of transcription of gene 7 in crown gall tumors. Transcription is different because the distance between the TATA box and the transcription initiation sites must be at least 40 nucleotides in yeast. Therefore, Saccharomyces cerevisiae does not appear to be a useful system for investigating the expression of T-DNA. Crown gall tumors were subjected to a number of stress agents, including heat shock, to determine the significance of the heat shock related sequence identified in gene 7. Primer extension analyses indicate that only cadmium and mercury have a significant effect on the expression of T-DNA gene 7. Although gene 7 responds to cadmium and mercury, the increase in transcription does not appear to be heat shock or metallothionein related, indicating that another mechanism is involved in the enhanced transcription of T-DNA gene 7 in crown gall tumors. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2010. / Committee Chair: Patricia Sobecky; Committee Member: Eric Stabb; Committee Member: Jim Spain; Committee Member: Roger Wartell; Committee Member: Thomas DiChristina. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Thesis (Ph.D.)--University of Adelaide, Dept. of Public Health, 2002. / Bibliography: p. 189-229.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [139]-158).

Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2008. / Committee Chair: Joseph Le Doux; Committee Member: Anthanassios Sambanis; Committee Member: David Archer; Committee Member: Michelle LaPlaca; Committee Member: Steve Stice; Committee Member: Todd McDevitt. Part of the SMARTech Electronic Thesis and Dissertation Collection.

Diss. (sammanfattning) Uppsala : Univ., 2001. / Härtill 4 uppsatser.

by Chan, Yuk Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 166-175). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.v / 摘要 --- p.vii / LIST OF CONTENTS --- p.viii / LIST OF FIGURES --- p.xiv / LIST OF TABLES --- p.xvii / LIST OF ABBREVIATIONS --- p.xviii / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.5 / Chapter 2.1 --- Vitamin A --- p.5 / Chapter 2.1.1 --- General and properties --- p.5 / Chapter 2.1.2 --- Biological importance of vitamin A --- p.6 / Chapter 2.1.3 --- Deficiency symptoms --- p.9 / Chapter 2.1.4 --- Dietary source of vitamin A --- p.10 / Chapter 2.1.5 --- Metabolism of dietary vitamin A and provitamin A in human --- p.12 / Chapter 2.1.5.1 --- Digestion and absorption --- p.12 / Chapter 2.1.5.2 --- Bioconversion --- p.12 / Chapter 2.1.5.2.1 --- "β, β-carotene-15,15'-monooxygenase (BCMO)" --- p.13 / Chapter 2.1.5.3 --- "Transport, uptake and storage" --- p.15 / Chapter 2.2 --- Vitamin A deficiency (VAD) --- p.19 / Chapter 2.2.1 --- Present situation --- p.19 / Chapter 2.2.2 --- Global efforts in dealing with VAD --- p.21 / Chapter 2.2.2.1 --- Vitamin A supplementation --- p.21 / Chapter 2.2.2.2 --- Food fortification --- p.22 / Chapter 2.2.2.3 --- Biofortification --- p.23 / Chapter 2.2.2.3.1 --- Conventional selective breeding --- p.23 / Chapter 2.2.2.3.2 --- Biosynthesis of provitamin A in plants --- p.25 / Chapter 2.2.2.3.3 --- Carotenoids enhancement in major plants and food crops --- p.31 / Chapter 2.3 --- Inherent problems of the present carotenoid enhancement --- p.34 / Chapter 2.3.1 --- Recommended Dietary Amount of vitamin A --- p.34 / Chapter 2.3.2 --- Factors affecting the bioefficacy of provitamin A in human body --- p.35 / Chapter 2.3.2.1 --- Bioavailability --- p.36 / Chapter 2.3.2.2 --- Bioconvertibility --- p.38 / Chapter 2.3.2.3 --- Health and nutrition status --- p.39 / Chapter 2.4 --- Previous study in our lab --- p.41 / Chapter 2.4.1 --- Overexpression of rice PSY1 --- p.41 / Chapter 2.4.2 --- Introduction of carotenoid genes and BCMOs into rice --- p.44 / Chapter 2.5 --- Overview of the project --- p.50 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.52 / Chapter 3.1 --- Chemicals --- p.52 / Chapter 3.2 --- Bacterial strains in regular cloning --- p.52 / Chapter 3.3 --- BCMO genes and carotenogenic genes --- p.53 / Chapter 3.4 --- Expression of BCMOs in bacterial system --- p.54 / Chapter 3.4.1 --- lac promoter system --- p.54 / Chapter 3.4.2 --- pBAD-TOPO® system --- p.56 / Chapter 3.5 --- Construction of gene cassettes for plant transformation --- p.58 / Chapter 3.5.1 --- Gene cassettes for carrot transformation --- p.58 / Chapter 3.5.1.1 --- Construction of gene cassettes for chicken or zebrafish bcmo driven by CaMV 35S promoter --- p.58 / Chapter 3.5.1.2 --- Construction of gene cassettes for chicken or zebrafish bcmo driven by lycopene-β-cyclase promoter --- p.63 / Chapter 3.5.2 --- Gene cassettes for Arabidopsis transformation --- p.67 / Chapter 3.5.2.1 --- Construction of gene cassettes expressing Dcpsy --- p.67 / Chapter 3.5.2.2 --- Construction of gene cassettes expressing mbcmos --- p.69 / Chapter 3.5.3 --- Gene cassettes for Rice transformation --- p.72 / Chapter 3.5.3.1 --- Construction of gene cassettes expressing mbcmos --- p.72 / Chapter 3.5.3.2 --- Construction of gene cassettes expressing Ospsyl and mbcmos --- p.74 / Chapter 3.5.4 --- Confirmation of sequence fidelity --- p.76 / Chapter 3.6 --- Carrot transformation --- p.76 / Chapter 3.6.1 --- Plant materials --- p.76 / Chapter 3.6.2 --- Preparation of Agrobacterium --- p.76 / Chapter 3.6.3 --- Agrobacterium mediated transformation --- p.77 / Chapter 3.6.3.1 --- Seed germination --- p.78 / Chapter 3.6.3.2 --- Co-cultivation with hypocotyls --- p.78 / Chapter 3.6.3.3 --- Callus induction and selection --- p.78 / Chapter 3.6.3.4 --- Liquid cell culture preparation and embryogenesis induction --- p.79 / Chapter 3.6.3.5 --- Regeneration --- p.80 / Chapter 3.7 --- Arabidopsis Transformation --- p.80 / Chapter 3.7.1 --- Plant materials --- p.80 / Chapter 3.7.2 --- Preparation of Agrobacterium --- p.81 / Chapter 3.7.3 --- Agrobacterium mediated transformation --- p.81 / Chapter 3.7.3.1 --- Co-cultivation --- p.81 / Chapter 3.7.3.2 --- Selection --- p.82 / Chapter 3.8 --- Rice transformation --- p.83 / Chapter 3.8.1 --- Plant materials --- p.83 / Chapter 3.8.2 --- Preparation of Agrobacterium --- p.83 / Chapter 3.8.3 --- Agrobacterium mediated transformation --- p.83 / Chapter 3.8.3.1 --- Callus induction from mature rice seeds --- p.84 / Chapter 3.8.3.2 --- Co-cultivation and selection --- p.84 / Chapter 3.9 --- Detection of transgene expression --- p.86 / Chapter 3.9.1 --- Detection at DNA level --- p.86 / Chapter 3.9.1.1 --- Genomic DNA extraction --- p.86 / Chapter 3.9.1.2 --- PCR screening --- p.86 / Chapter 3.9.1.3 --- Synthesis of DIG-labelled DNA probes --- p.86 / Chapter 3.9.1.4 --- Southern blot analysis --- p.87 / Chapter 3.9.2 --- Detection at RNA level --- p.88 / Chapter 3.9.2.1 --- Total RNA extraction --- p.88 / Chapter 3.9.2.2 --- Northern blot analysis --- p.89 / Chapter 3.9.2.3 --- RT-PCR --- p.89 / Chapter 3.9.3 --- Detection at protein level --- p.89 / Chapter 3.9.3.1 --- Antibody production --- p.89 / Chapter 3.9.3.1.1 --- B.CMO protein induction in pET30a-bacterial system --- p.90 / Chapter 3.9.3.1.2 --- Immunization of rabbit and serum collection --- p.93 / Chapter 3.9.3.2 --- Protein extraction and Tricine SDS-PAGE --- p.93 / Chapter 3.9.3.3 --- Western blot analysis --- p.94 / Chapter 3.9.4 --- Detection at final product level --- p.95 / Chapter 3.9.4.1 --- UPLC analysis --- p.95 / Chapter 3.9.4.1.1 --- Extraction of total carotenoids and retinoids --- p.95 / Chapter 3.9.4.1.2 --- UPLC identification --- p.96 / Chapter CHAPTER 4. --- RESULTS --- p.97 / Chapter 4.1 --- Modified bcmo genes --- p.97 / Chapter 4.2 --- Expression of BCMOs in bacterial system --- p.102 / Chapter 4.2.1 --- lac promoter system --- p.104 / Chapter 4.2.2 --- pBAD-TOPO® system --- p.106 / Chapter 4.2.3 --- UPLC detection --- p.108 / Chapter 4.3 --- Carrot transformation --- p.110 / Chapter 4.3.1 --- Construction of gene cassettes for carrot transformation --- p.110 / Chapter 4.3.2 --- Seed germination and co-cultivation --- p.112 / Chapter 4.3.3 --- Callus induction and selection --- p.113 / Chapter 4.3.4 --- Embryogenesis induction and regeneration --- p.113 / Chapter 4.3.5 --- Callus induction in the dark --- p.115 / Chapter 4.3.6 --- Detection of native BCMO --- p.116 / Chapter 4.3.6.1 --- Genomic PCR screening of 35Spro - zebcmo transgenic lines --- p.116 / Chapter 4.3.6.2 --- Southern blot analysis of 35Spro - zebcmo transgenic lines --- p.117 / Chapter 4.3.6.3 --- RT-PCR of 35Spro - zebcmo transgenic lines --- p.118 / Chapter 4.3.6.4 --- Detection at protein level --- p.119 / Chapter 4.3.6.4.1 --- Antibody production --- p.119 / Chapter 4.3.6.5 --- Western blot analysis of 35Spro - zebcmo transgenic lines --- p.123 / Chapter 4.3.6.6 --- Genomic PCR screening of later transgenic lines --- p.123 / Chapter 4.3.6.7 --- Western blot analysis of later transgenic lines --- p.125 / Chapter 4.3.6.8 --- UPLC analysis of later transgenic lines --- p.127 / Chapter 4.3.7 --- Detection of modified BCMO --- p.130 / Chapter 4.3.7.1 --- Genomic PCR screening --- p.130 / Chapter 4.3.7.2 --- Northern blot analysis --- p.132 / Chapter 4.3.7.3 --- Western blot analysis --- p.134 / Chapter 4.3.8 --- UPLC analysis --- p.136 / Chapter 4.4 --- Arabidopsis transformation --- p.138 / Chapter 4.4.1 --- Construction of gene cassettes for Arabidopsis transformation --- p.138 / Chapter 4.4.2 --- Selection --- p.139 / Chapter 4.4.3 --- Genmoic PCR screening of Arabidopsis transformants --- p.140 / Chapter 4.4.4 --- UPLC analysis for Arabidopsis transformants --- p.142 / Chapter 4.5 --- Rice transformation --- p.144 / Chapter 4.5.1 --- Construction of gene cassettes for rice transformation --- p.144 / Chapter 4.5.2 --- "Callus induction from mature rice seeds, co-cultivation and selection" --- p.146 / Chapter 4.5.3 --- Genomic PCR screening of Rice transformants --- p.147 / Chapter 4.5.4 --- UPLC analysis of rice transformants --- p.149 / Chapter CHAPTER 5. --- DISCUSSION --- p.151 / Chapter 5.1 --- Bacterial expression of BCMO --- p.151 / Chapter 5.2 --- Analysis of BCMO in plants --- p.153 / Chapter 5.2.1 --- Carrot --- p.154 / Chapter 5.2.1.1 --- Expression of BCMO in carrot transformants --- p.154 / Chapter 5.2.1.2 --- UPLC analysis of carrot transformants --- p.155 / Chapter 5.2.2 --- Arabidopsis --- p.156 / Chapter 5.2.3 --- Rice --- p.158 / Chapter 5.3 --- Proposed explanation for the failure of retinal production --- p.159 / Chapter 5.3.1 --- Retinal sequestration --- p.160 / Chapter 5.3.2 --- Localization of BCMO --- p.161 / Chapter 5.4 --- Future prospects --- p.163 / Chapter CHAPTER 6. --- CONCLUSIONS --- p.165 / REFERENCES --- p.166 / APPENDICES --- p.176

by Lee Juon Kiu. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 139-152). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / Table of contents --- p.ix / List of figures --- p.xv / List of tables --- p.xvii / List of graphs --- p.xviii / List of abbreviations --- p.xix / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter Chapter 2: --- Literature Review --- p.4 / Chapter 2.1 --- Human granulocyte colony-stimulating factor (hG-CSF) --- p.4 / Chapter 2.1.1 --- Physiological roles --- p.4 / Chapter 2.1.2 --- Molecular properties --- p.8 / Chapter 2.1.3 --- Biochemical properties --- p.9 / Chapter 2.1.4 --- Comparison to G-CSF of other specie --- p.10 / Chapter 2.1.5 --- Clinical application --- p.11 / Chapter 2.1.6 --- Economic value --- p.13 / Chapter 2.2 --- Expression systems producing recombinant hG-CSF --- p.15 / Chapter 2.2.1 --- Bacteria --- p.15 / Chapter 2.2.2 --- Yeasts --- p.17 / Chapter 2.2.3 --- Animal cell lines --- p.18 / Chapter 2.2.4 --- Transgenic animals --- p.19 / Chapter 2.2.5 --- Transgenic plants --- p.20 / Chapter 2.3 --- Plant as bioreactors --- p.21 / Chapter 2.3.1 --- Characteristics of using plant as bioreactors --- p.22 / Chapter 2.3.2 --- Transgenic plants producing hematopoietic growth factors --- p.24 / Chapter 2.3.2.1 --- Granulocyte-macrophage colony-stimulating factor (GM-CSF) --- p.24 / Chapter 2.3.2.2 --- Erythropoietin (Epo) --- p.26 / Chapter 2.3.3 --- Arabidopsis and tobacco as model plants --- p.27 / Chapter 2.3.3.1 --- Arabidopsis --- p.28 / Chapter 2.3.3.2 --- Tobacco --- p.28 / Chapter 2.3.4 --- Phaseolin and its regulatory sequences --- p.29 / Chapter 2.4 --- Plant transformation methods --- p.31 / Chapter 2.4.1 --- Agrobacterium-mediated transformation --- p.31 / Chapter 2.4.1.1 --- Tissue culture methods --- p.31 / Chapter 2.4.1.2 --- Non-tissue culture (In planta) methods --- p.32 / Chapter 2.4.2 --- Direct DNA uptake transformation --- p.33 / Chapter 2.4.2.1 --- Chemical methods --- p.33 / Chapter 2.4.2.2 --- Electrical methods --- p.34 / Chapter 2.4.2.3 --- Physical methods --- p.34 / Chapter Chapter 3: --- Materials and Methods --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.2 --- Chemicals --- p.37 / Chapter 3.3 --- Bacterial strains --- p.37 / Chapter 3.4 --- Chimeric gene construction --- p.37 / Chapter 3.4.1 --- Cloning of pTZ/Phas/His/EK/hG-CSF --- p.41 / Chapter 3.4.2 --- Cloning of pBK/Phas/SP/His/EK/hG-CSF --- p.44 / Chapter 3.4.3 --- Cloning of pBK/Phas/SP/hG-CSF --- p.47 / Chapter 3.4.4 --- Confirmation of sequence fidelity of chimeric genes --- p.50 / Chapter 3.4.5 --- Cloning of chimeric genes into Agrobacterium binary vector --- p.51 / Chapter 3.5 --- Expression in Arabidopsis --- p.52 / Chapter 3.5.1 --- Agrobacterium GV3101/pMP90 transformation --- p.52 / Chapter 3.5.2 --- Arabidopsis transformation --- p.53 / Chapter 3.5.2.1 --- Plant materials --- p.53 / Chapter 3.5.2.2 --- Vacuum infiltration --- p.54 / Chapter 3.5.3 --- Screening of successful R1 transformants --- p.55 / Chapter 3.5.4 --- Screening of hemizygous and homozygous transgenic Arabidopsis --- p.56 / Chapter 3.5.5 --- GUS assay --- p.57 / Chapter 3.5.6 --- Genomic DNA extraction --- p.57 / Chapter 3.5.7 --- Southern blot analysis --- p.58 / Chapter 3.5.8 --- Total RNA extraction from developing siliques --- p.59 / Chapter 3.5.9 --- Northern blot analysis --- p.60 / Chapter 3.5.10 --- Protein extraction and Tricine SDS-PAGE --- p.61 / Chapter 3.5.11 --- Western blot analysis --- p.62 / Chapter 3.5.12 --- Functional analysis --- p.63 / Chapter 3.5.12.1 --- Culture ofNFS-60 cells --- p.64 / Chapter 3.5.12.2 --- MTT assay --- p.65 / Chapter 3.6 --- Expression in tobacco --- p.67 / Chapter 3.6.1 --- Agrobacterium LBA4404/pAL4404 transformation --- p.67 / Chapter 3.6.2 --- Tobacco transformation --- p.68 / Chapter 3.6.2.1 --- Plant materials --- p.68 / Chapter 3.6.2.2 --- Tobacco transformation using leaf-disc technique --- p.68 / Chapter 3.6.3 --- Regeneration of transgenic tobacco --- p.69 / Chapter 3.6.4 --- GUS assay --- p.70 / Chapter 3.6.5 --- Genomic DNA extraction --- p.70 / Chapter 3.6.6 --- Southern blot analysis --- p.70 / Chapter 3.6.7 --- Total RNA extraction from immature seeds --- p.70 / Chapter 3.6.8 --- Northern blot analysis --- p.71 / Chapter 3.6.9 --- Protein extraction and Tricine SDS-PAGE --- p.71 / Chapter 3.6.10 --- Western blot analysis --- p.71 / Chapter 3.6.11 --- Functional analysis --- p.71 / Chapter 3.6.11.1 --- Culture of NFS-60 cells --- p.72 / Chapter 3.6.11.2 --- MTT assay --- p.72 / Chapter Chapter 4: --- Results --- p.73 / Chapter 4.1 --- Chimeric gene construction --- p.73 / Chapter 4.1.1 --- Cloning of pTZ/Phas/His/EK/hG-CSF --- p.73 / Chapter 4.1.2 --- Cloning of pBK/Phas/SP/His/EK/hG-CSF --- p.75 / Chapter 4.1.3 --- Cloning of pBK/Phas/SP/hG-CSF --- p.77 / Chapter 4.1.4 --- Cloning of chimeric genes into Agrobacterium binary vector --- p.79 / Chapter 4.2 --- Expression in Arabidopsis --- p.81 / Chapter 4.2.1 --- Agrobacterium GV3101/pMP90 transformation --- p.81 / Chapter 4.2.2 --- Arabidopsis transformation and screening of R1 transformants --- p.83 / Chapter 4.2.3 --- Screening of hemizygous transgenic R1 Arabidopsis --- p.84 / Chapter 4.2.4 --- Screening of homozygous transgenic R2 Arabidopsis --- p.86 / Chapter 4.2.5 --- GUS assay --- p.88 / Chapter 4.2.6 --- Genomic DNA extraction --- p.89 / Chapter 4.2.7 --- Southern blot analysis --- p.91 / Chapter 4.2.8 --- Total RNA extraction from developing siliques --- p.93 / Chapter 4.2.9 --- Northern blot analysis --- p.94 / Chapter 4.2.10 --- Protein extraction and Tricine SDS-PAGE --- p.96 / Chapter 4.2.11 --- Western blot analysis --- p.99 / Chapter 4.2.12 --- Functional analysis --- p.103 / Chapter 4.3 --- Expression in tobacco --- p.108 / Chapter 4.3.1 --- Agrobacterium LBA4404/pAL4404 transformation --- p.108 / Chapter 4.3.2 --- Tobacco transformation and regeneration of transformants --- p.109 / Chapter 4.3.3 --- GUS assay --- p.111 / Chapter 4.3.4 --- Genomic DNA extraction --- p.112 / Chapter 4.3.5 --- Southern blot analysis --- p.114 / Chapter 4.3.6 --- Total RNA extraction from immature seeds --- p.116 / Chapter 4.3.7 --- Northern blot analysis --- p.116 / Chapter 4.3.8 --- Protein extraction and Tricine SDS-PAGE --- p.118 / Chapter 4.3.9 --- Western blot analysis --- p.120 / Chapter 4.3.10 --- Functional analysis --- p.123 / Chapter Chapter 5: --- Discussion --- p.126 / Chapter 5.1 --- Introduction --- p.126 / Chapter 5.2 --- Successful in producing biologically active rhG-CSF from transgenic plants --- p.128 / Chapter 5.2.1 --- Production level --- p.129 / Chapter 5.2.2 --- O-glycosylation --- p.130 / Chapter 5.2.3 --- Phaseolin signal peptide --- p.131 / Chapter 5.2.4 --- Functional analysis --- p.131 / Chapter 5.3 --- Comparison of the productivity of other expression systems producing rhG-CSF --- p.132 / Chapter 5.4 --- Comparison of the productivity of plants producing different human proteins --- p.135 / Chapter 5.5 --- Future perspectives --- p.137 / Chapter Chapter 6: --- Conclusion --- p.138 / References --- p.139