No description available.

Thesis(M.S.)--Auburn University, 2005. / Abstract. Vita. Includes bibliographic references.

No description available.

No description available.

Thesis (Ph. D.)--Auburn University. / Abstract. Vita. Includes bibliographical references (p. 66-71).

by Henry, Kam Yin Cheung. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 148-160). / ZEBRAFISH (BRACHYDANIO RERIO) / ACKNOWLEDGEMENTS / LIST OF CONTENTS / ABSTRACT / ABBREVIATION / Chapter CHAPTER ONE --- INTRODUCTION / Chapter 1.1 --- Transgenic fish --- p.1 / Chapter 1.2 --- Zebrafish --- p.4 / Chapter 1.3 --- The grass carp GH gene and protein / Chapter 1.3.1 --- The genomic sequence --- p.5 / Chapter 1.3.2 --- The cDNA sequence --- p.7 / Chapter 1.3.3 --- The grass carp GH protein --- p.7 / Chapter 1.4 --- Functional aspects of promoter regions / Chapter 1.4.1 --- PEPCK --- p.9 / Chapter 1.4.2 --- RSV-LTR --- p.10 / Chapter 1.4.3 --- hMT-IIA --- p.10 / Chapter 1.4.4 --- MMTV-LTR --- p.11 / Chapter 1.5 --- Eukaryotic gene expression in cultured cells / Chapter 1.5.1 --- COS-7 and HepG2 cells --- p.11 / Chapter 1.5.2 --- Transfection system --- p.12 / Chapter 1.5.3 --- Fate of DNA after transfection --- p.13 / Chapter 1.6 --- Electroporation and microinjection as tools for gene transfer / Chapter 1.6.1 --- Electroporation: Theory and operation --- p.13 / Chapter 1.6.2 --- Microinjection: Design of microinjector --- p.16 / Chapter 1.6.3 --- Fate of DNA after gene transfer in embryos / Transient expression --- p.16 / Stable transformation --- p.17 / Inheredity of transgene --- p.17 / Chapter 1.7 --- The aims of the present study --- p.18 / Chapter CHAPTER TWO --- MATERIALS AND METHODS / Chapter 2.1 --- General techniques / Chapter 2.1.1 --- Electrophoresis of DNA / Chapter 2.1.1.1 --- Agarose gel electrophoresis --- p.19 / Chapter 2.1.1.2 --- PAGE --- p.20 / Chapter 2.1.2 --- Purification of DNA --- p.21 / Chapter 2.1.3 --- Recovery of DNA fragments / Chapter 2.1.3.1 --- Electroelution --- p.22 / Chapter 2.1.3.2 --- Geneclean kit --- p.23 / Chapter 2.1.4 --- Standard recombinant DNA techniques / Chapter 2.1.4.1 --- Dephosphorylation --- p.24 / Chapter 2.1.4.2 --- Kinasing --- p.24 / Chapter 2.1.4.3 --- Ligation --- p.24 / Chapter 2.1.4.4 --- Filling in reaction --- p.25 / Chapter 2.1.4.5 --- Transformation --- p.25 / Chapter 2.1.5 --- Minipreparation of plasmids --- p.26 / Chapter 2.1.6 --- Large preparation of plasmids / Chapter 2.1.6.1 --- Qiagene kit --- p.27 / Chapter 2.1.6.2 --- CsCl density gradient centrifugation --- p.27 / Chapter 2.1.7 --- DNA sequencing --- p.29 / Chapter 2.1.8 --- "Extraction of DNA from embryos, fry and fish" / Method 1 --- p.32 / Method 2 --- p.32 / Chapter 2.1.9 --- Probe labelling / Chapter 2.1.9.1 --- End-labelling --- p.33 / Chapter 2.1.9.2 --- Random priming --- p.33 / Chapter 2.1.10 --- CAT assay --- p.33 / Chapter 2.1.11 --- Polymerase chain reaction(PCR) --- p.35 / Chapter 2.1.12 --- Radioimmunassay(RIA) of FGH --- p.36 / Chapter 2.1.13 --- Dot blotting --- p.38 / Chapter 2.1.14 --- Southern blotting --- p.39 / Chapter 2.2 --- "Linkers, primers and probes" / Chapter 2.2.1 --- Primers --- p.41 / Chapter 2.2.2 --- Linkers --- p.45 / Chapter 2.2.3 --- Probes --- p.47 / Chapter 2.3 --- Construction of chimeric growth hormone genes / Chapter 2.3.1 --- Sources of plasmids --- p.50 / Chapter 2.3.2 --- General principles --- p.50 / Chapter 2.3.3 --- PEPCKgcGHcDNA --- p.51 / Chapter 2.3.4 --- RSVgcGHcDNA --- p.54 / Chapter 2.3.5 --- hMTgcGHcDNAcDNA --- p.56 / Chapter 2.3.6 --- MMTVgcGHcDNA --- p.58 / Chapter 2.3.7 --- "PEPCKgcGH, RSVgcGH and hMTgcGH" --- p.60 / Chapter 2.4 --- Expression of chimeric genes in cultured cells / Chapter 2.4.1 --- Culturing of COS-7 and HepG2 cells --- p.66 / Chapter 2.4.2 --- Expression of chimeric genes in COS-7 and HepG2 cells --- p.67 / Chapter 2.5 --- Zebrafish / Chapter 2.5.1 --- "Culturing, Spawning and hatching" --- p.67 / Chapter 2.6 --- Electroporation and microinjection for gene transfer / Chapter 2.6.1 --- Electroporation / Chapter 2.6.1.1 --- Tuning up electroporation --- p.69 / Chapter 2.6.1.2 --- Evidence of gene transfer by electroporation / Chapter 2.6.1.2.1 --- CAT assay --- p.71 / Chapter 2.6.1.2.2 --- Dot blot --- p.71 / Chapter 2.6.1.2.3 --- PCR and Southern blotting of PCR products --- p.72 / Chapter 2.6.1.2.4 --- Southern blotting of fish total DNA --- p.73 / Chapter 2.6.2 --- Microinjection / Chapter 2.6.2.1 --- Handling of microinjection --- p.74 / Chapter 2.6.2.2 --- Evidence of gene transfer by microinjection / Chapter 2.6.2.2.1 --- CAT assay --- p.75 / Chapter 2.6.2.2.2 --- PCR and Southern blotting of PCR products --- p.75 / Chapter 2.7 --- Phenotypic alteration of fish generated from electroporated eggs / Chapter 2.7.1 --- Electroporation and handling of fish generated from electroporation --- p.75 / Chapter 2.7.2 --- Measurement of phenotypic change in fish generated from electroporation --- p.77 / Chapter 2.8 --- Detection of transgene and expression of exogenous DNA / Chapter 2.8.1 --- Transgene detection --- p.78 / Chapter 2.8.2 --- Expression of exogenous DNA --- p.79 / Chapter CHAPTER THREE --- RESULTS / Chapter 3.1 --- Construction of Chimeric growth hormone genes / Chapter 3.1.1 --- Confirmation of integrity of chimeric genes / PEPCKgcGHcDNA --- p.80 / RSVgcGHcDNA --- p.81 / hMTgcGHcDNA --- p.81 / MMTVgcGHcDNA --- p.81 / "PEPCKgcGH, RSVgcGH and hMTgcGH" --- p.82 / Chapter 3.1.2 --- Yield of chimeric genes from CsCl density gradient centrifugation --- p.82 / Chapter 3.2 --- Chimeric gene expression in COS-7 and HepG2 cells / Chapter 3.2.1 --- Expression of chimeric genes in COS-7 cells --- p.89 / Chapter 3.2.2 --- Expression of chimeric genes in HepG2 cells --- p.93 / Chapter 3.3 --- Transfer of chimeric genes into embryos / Chapter 3.3.1 --- Electroporation / Chapter 3.3.1.1 --- Monitoring of electroporation --- p.94 / Chapter 3.3.1.2 --- Evidence for gene transfer / Chapter 3.3.1.2.1 --- CAT assay --- p.98 / Chapter 3.3.1.2.2 --- Dot blotting --- p.98 / Chapter 3.3.1.2.3 --- PCR and Southern blotting of PCR product --- p.101 / Chapter 3.3.1.2.4 --- Southern blotting of DNA from fish generated from electroporation --- p.106 / Chapter 3.3.2 --- Microinjection / Chapter 3.3.2.1 --- CAT assay --- p.109 / Chapter 3.3.2.2 --- PCR --- p.109 / Chapter 3.4 --- Phenotypic alterations of fish / The first experiment --- p.112 / The second experiment --- p.113 / The third experiment --- p.113 / The fourth experiment --- p.122 / Chapter 3.5 --- Detection of transgene and expression of exogenous DNA / Chapter 3.5.1 --- Transgene --- p.128 / Chapter 3.5.2 --- Possible expression of exogenous DNA --- p.129 / Chapter CHAPTER FOUR --- DISCUSSION / Chapter 4.1 --- Chimeric growth hormone genes --- p.132 / Chapter 4.2 --- Expression of chimeric growth hormone genes in COS-7 and HepG2 cells --- p.134 / Chapter 4.3 --- Transfer of exogenous DNA into embyros --- p.136 / Chapter 4.4 --- Phenotypic alteration of fish developed from electroporated eggs --- p.139 / Chapter 4.5 --- The possible integration and expression of exogenous DNA --- p.143 / Chapter 4.6 --- Conclusions --- p.145 / Chapter 4.7 --- Suggestions for further studies --- p.146 / REFERENCES --- p.148 / Chapter APPENDIX I --- Restriction maps / PEPCKgcGH / PEPCKgcGHcDNA / RSVgcGH / RSVgcGHcDNA / hMTgcGH / hMTgcGHcDNA / MMTVgcGHcDNA / pBH1.2 / pMSG-CAT / pUC19 / hMT-IIA / PBC12BI / RSVCAT / pUC101 / pSEl/S2 / PUCSE2/S1 / pUCS2

Preliminary analyses of three kinds of promoter activity showed that a-actin gene promoter was chosen to initiate the hormone transcription for the first consideration. We have fused the cDNAs encoding the intact somatolactins in frame to a zebrafish a-actin gene promoter to generate transgenic zebrafish lines co-injected with a GFP protein driven by the same promoter. The transgenic zebrafish were selected from GFP expression and confirmed by genomic PCR and Southern blot analysis, then maintained as transgenic founders. Measurement of the transgenes' expressions and the expressions of marker genes in different pathways by using real-time PCR provided a general understanding of SLs' actions. The data obtained indicated that the over-expressing of SLalpha and SLbeta in vivo significantly enhance the transcriptions of the insulin-like growth factors, IGF1 (5.46-fold and 6.77-fold), IGF2a (4.38-fold and 4.35-fold) and IGF2b (2.83-fold and 3.94-fold), but down-regulated IGF3 (a novel member found specifically in gonad) in larvae. However, the stimulation by administration of recombinant proteins (SLalpha and SLbeta) only showed a slight induction of the mRNA levels of IGFs (IGF1, IGF2a and IGF2b) on ZFL cells in vitro. / Somatolactin (SL) is a novel member of pituitary polypeptide hormone found only in fish; it shares significant structural homology with prolactin and growth hormone. Since somatolactin receptor (SLR) was first defined as GHR1 and orthologous to the growth hormone receptor GHR2, SL and GH may share similar actions in growth and development. Recently, two SLs have been identified as SLalpha and SLbeta with similar structures, freshwater fish have these two isoforms found in the same species and only one isoform (SLalpha) is found in marine species. The two isoforms of SL may have different functions and physiological actions. To investigate the roles of SLs on vertebrate development and embryogenesis, we generated transgenic fish models with "all zebrafish" elements in origin to study the physiological functions of SLs in zebrafish. / The ectopic expression of somatolactins also results in up-regulating gene expression of insulin, leptin, sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FAS), as well as the expression of vitellogenin and melanocyte-stimulating hormone (MSH) levels while causing reduction of catalase (CAT) and glutathione S-transferase (GST) levels in larvae. The results here represent the similar function between SLalpha and SLbeta and reveal more details in fish of the endocrinology system involvement in growth development, glucose synthesis, lipid metabolism, reproduction, pigmentation and antioxidant defense system through the actions of SLs. / Three different gene promoters of zebrafish have been isolated to initiate the ectopic expression of somatolactins in vivo, which including a constitutional beta-actin gene promoter, a liver specific transferrin gene promoter and a zinc ion inducible metallothionein (MT) gene promoter. The promoter activities were tested in fish cell-line by using luciferase reporter assay. In MT gene promoter, two alleles of a zebrafish metallothionein II gene (zMT-II) promoter (zMT-IIA and zMT-IIB) containing 10 MREs in the 5'-flanking region (1,514 bp) were identified in zebrafish. These putative MREs were confirmed via electrophoretic mobility shift assay (EMSA) to have binding activities from the cellular and nuclear extracts of a zebrafish cell line, ZFL. Transient gene expression studies using zebrafish liver (ZFL) cell lines also confirmed that the most distal cluster of MREs contributed to the maximal induction of zMT-IIA activity by Zn2+ and the Zn 2+ induction was dose-dependent. EMSA also identified transcription factor(s) of two different sizes from the cytoplasmic and nuclear extracts of the ZFL cells that were able to bind with the MREs, but no increase in MRE binding was detected in the extracts of these cells after Zn2+ or Cd2+ treatment, compared with untreated control cells. The mechanisms of MT gene transcription induction via metal ions are discussed herein. / Wan, Guohui. / Adviser: Chan King Ming. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 139-163). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2005. / Bibliography: leaves 62-69.

by Yan Hiu Mei, Carol. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 114-126). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.v / List of Tables --- p.viii / List of Figures --- p.ix / Abbreviations --- p.xii / Chapter CHAPTER 1 --- TRANSGENIC TECHNOLOGY --- p.1 / Chapter 1.1 --- Transgenesis in animals --- p.1 / Chapter 1.2 --- Transgenic fish in toxicology --- p.4 / Chapter 1.2.1 --- Aquatic metal toxicity --- p.4 / Chapter 1.2.2 --- Environmental monitoring of aquatic metal toxicity --- p.5 / Chapter 1.2.3 --- Biomarkers --- p.6 / Chapter 1.3 --- Transgenics in aquaculture --- p.9 / Chapter 1.3.1 --- Revolution is needed in aquaculture --- p.9 / Chapter 1.3.2 --- Aquaculture potential of tilapia in China --- p.10 / Chapter 1.3.3 --- Endocrinology for fish growth --- p.12 / Chapter 1.3.4 --- Growth promotion by exogenous growth hormone in tilapia --- p.14 / Chapter 1.3.5 --- Accelerated growth in transgenic fish --- p.15 / Chapter 1.4 --- General principle in transgenic fish production --- p.16 / Chapter 1.5 --- Project aim --- p.22 / Chapter CHAPTER 2 --- ISOLATION AND CHARACTERIZATION OF ZEBRAFISH METALLOTHIONEIN GENE PROMOTER --- p.23 / Chapter 2.1 --- Introduction --- p.23 / Chapter 2.1.1 --- Metallothionein --- p.23 / Chapter 2.1.2 --- Biological functions --- p.24 / Chapter 2.1.3 --- Metallothionein gene regulations --- p.25 / Chapter 2.1.4 --- Metallothionein as biomarker for metal pollution --- p.26 / Chapter 2.2 --- Materials and methods --- p.28 / Chapter 2.2.1 --- General molecular biology techniques --- p.28 / Chapter 2.2.2 --- Sequences of PCR primers used --- p.31 / Chapter 2.2.3 --- Cloning zebrafish MT gene 5-flanking region --- p.31 / Chapter 2.2.4 --- Cloning zebrafish MT gene --- p.32 / Chapter 2.2.5 --- Cloning full length zMT gene --- p.33 / Chapter 2.2.6 --- Cell culture --- p.35 / Chapter 2.2.7 --- Transient transfection assay --- p.37 / Chapter 2.2.8 --- Electrophoretic mobility shift assay --- p.39 / Chapter 2.3 --- Results --- p.42 / Chapter 2.3.1 --- Zebrafish metallothionein gene --- p.42 / Chapter 2.3.2 --- Deletion analysis of zMT promoter by transient transfection assay --- p.48 / Chapter 2.3.3 --- Functional characterization of zebrafish metallothionein promoter --- p.57 / Chapter 2.4 --- Discussions --- p.61 / Chapter 2.4.1 --- Zebrafish MT gene --- p.61 / Chapter 2.4.2 --- Functional characterization of zebrafish MT promoter --- p.61 / Chapter CHAPTER 3 --- PREPARATION OF GENE CONSTRUCTS FOR TRANSFER IN ZEBRAFISH --- p.65 / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.1.1 --- Zebrafish as model in toxicological studies --- p.65 / Chapter 3.1.2 --- Reporter gene system --- p.66 / Chapter 3.1.3 --- Transgenic reporter fish --- p.68 / Chapter 3.1.4 --- Gene transfer by electroporation in zebrafish --- p.68 / Chapter 3.1.5 --- Objective --- p.69 / Chapter 3.2 --- Materials and methods --- p.70 / Chapter 3.2.1 --- Design of gene constructs for ectopic expression in zebrafish --- p.70 / Chapter 3.2.2 --- Testing electroporation conditions for zebrafish --- p.72 / Chapter 3.3 --- Results --- p.73 / Chapter 3.4 --- Discussions --- p.76 / Chapter 3.4.1 --- Engineering gene constructs --- p.76 / Chapter 3.4.2 --- Applications of transgenic zebrafish --- p.79 / Chapter CHAPTER 4 --- GENE TRANSFER EXPERIMENTS ON TILAPIA --- p.82 / Chapter 4.1 --- Introduction --- p.82 / Chapter 4.2 --- Materials and methods --- p.85 / Chapter 4.2.1 --- Isolation of O. aureus growth hormone --- p.85 / Chapter 4.2.2 --- Engineering gene constructs for ectopic expression in tilapia --- p.86 / Chapter 4.2.3 --- Gene transfer in tilapia --- p.87 / Chapter 4.2.4 --- Screening transgenic tilapia --- p.89 / Chapter 4.3 --- Results --- p.91 / Chapter 4.3.1 --- Tilapia growth hormone --- p.91 / Chapter 4.3.2 --- Gene constructs for ectopic expression in tilapia --- p.94 / Chapter 4.3.3 --- Testing electroporation conditions --- p.96 / Chapter 4.3.4 --- PCR screening for transgenic fish --- p.97 / Chapter 4.4 --- Discussions --- p.101 / Chapter 4.4.1 --- Tilapia growth hormone --- p.101 / Chapter 4.4.2 --- Electroporation experiments on of tilapia eggs --- p.101 / Chapter 4.4.3 --- Improvements on gene construct design for tilapia --- p.104 / Chapter 4.4.4 --- Ethical and safety considerations --- p.106 / Chapter CHAPTER 5 --- REFERENCES --- p.114 / APPENDIX --- p.127