Factor VIII is a clotting factor found on the intrinsic side of the coagulation cascade. A mutation in the factor VIII gene causes the disease Hemophilia A, for which there is no cure. The most common treatment is administration of recombinant factor VIII. However, this can cause an immune response that renders the treatment ineffective in certain hemophilia patients. For this reason a new treatment, or cure, needs to be developed. Gene editing is one solution to correcting the factor VIII mutation. CRISPR/Cas9 mediated gene editing introduces a double stranded break in the genomic DNA. Where this break occurs repair mechanisms cause insertions and deletions, or if a template oligonucleotide can be provided point mutations could be introduced or corrected. However, to accomplish this goal for editing factor VIII mutations, a way to deliver the components of CRISPR/Cas9 into somatic cells is needed. In this study, I confirmed that the CRISPR/Cas9 system was able to create a mutation in the factor VIII gene in zebrafish. I also showed that the components of CRISPR/Cas9 could be piggybacked by vivo morpholino into a variety of blood cells. This study also confirmed that the vivo morpholino did not interfere with the gRNA binding to the DNA, or Cas9 protein inducing the double stranded break.

In the present study, the ease of rearing, short generation time and molecular research tools available for the zebrafish model (Danio rerio, Hamilton) were exploited to investigate transcriptional regulation in relation to feeding, photoperiod and experimental selection. Chapter 2 describes transcriptional regulation in fast skeletal muscle following fasting and a single satiating meal of bloodworms. Changes in transcript abundance were investigated in relation to the food content in the gut. Using qPCR, the transcription patterns of 16 genes comprising the insulin-like growth factor (IGF) system were characterized, and differential regulation between some of the paralogues was recorded. For example, feeding was associated with upregulation of igf1a and igf2b at 3 and 6h after the single-meal was offered, respectively, whereas igf1b was not detected in skeletal muscle. On the other hand, fasting triggered the upregulation of the igf1 receptors and igfbp1a/b, the only binding proteins whose transcription was responsive to a single-satiating meal. In addition to the investigation of the IGF-axis, an agnostic approach was used to discover other genes involved in transcriptional response to nutritional status, by employing a whole-genome microarray containing 44K probes. This resulted in the discovery of 147 genes in skeletal muscle that were differentially expressed between fasting and satiation. Ubiquitin-ligases involved in proteasome-mediated protein degradation, and antiproliferative and pro-apoptotic genes were among the genes upregulated during fasting, whereas satiation resulted in an upregulation of genes involved in protein synthesis and folding, and a gene highly correlated with growth in mice and fish, the enzyme ornithine decarboxylase 1. Zebrafish exhibit circadian rhythms of breeding, locomotor activity and feeding that are controlled by molecular clock mechanisms in central and peripheral organs. In chapter 3 the transcription of 17 known clock genes was investigated in skeletal muscle in relation to the photoperiod and food content in the gut. The hypothesis that myogenic regulatory factors and components of the IGF-pathway were clock-controlled was also tested. Positive (clock1 and bmal1 paralogues) and negative oscillators (cry1a and per genes) showed a strong circadian pattern in skeletal muscle in anti-phase with each other. MyoD was not clock-controlled in zebrafish in contrast to findings in mice, whereas myf6 showed a circadian pattern of expression in phase with clock and bmal. Similarly, the expression of two IGF binding proteins (igfbp3 and 5b) was circadian and in phase with the positive oscillators clock and bmal. It was also found that some paralogues responded differently to photoperiod. For example, clock1a was 3-fold more responsive than clock1b. Cry1b did not show a circadian pattern of expression. These patterns of expression provide evidence that the molecular clock mechanisms in skeletal muscle are synchronized with the molecular clock in central pacemaker organs such as eyes and the pineal gland. Using the short generation time of zebrafish the effects of selective breeding for body size at age were investigated and are described in chapter 4. Three rounds of artificial selection for small (S-lineage) and large body size (L-lineage) resulted in zebrafish populations whose average standard length were, respectively, 2% lower and 10% higher than an unselected control lineage (U-lineage). Fish from the L-lineage showed an increased egg production and bigger egg size with more yolk, possibly contributing to the larger body size observed in the early larval stage (6dpf) of fish from this lineage. Fish from S- and L-lineage exposed to fasting and refeeding showed very similar feed intake, providing evidence that experimental selection did not cause significant changes in appetite control. Investigation of the expression of the IGF-axis and nutritionally-response in skeletal muscle after fasting and refeeding revealed that the pattern of expression was not different between the selected lineages, but that a differential responsiveness was observed in a limited number of genes, providing evidence that experimental selection might have changed the way fish allocate the energy acquired through feeding. For example, a constitutive higher expression of igf1a was recorded in skeletal muscle of fish from the L-lineage whereas igfbp1a/b transcripts were higher in muscle of fish from the S-lineage. These findings demonstrate the rapid changes in growth and transcriptional response in skeletal muscle of zebrafish after only three rounds of selection. Furthermore, it provides evidences that differences in growth during embryonic and larval stages might be related to higher levels of energy deposited during oogenesis, whereas differences in adult fish were better explained by changes in energy allocation instead of energy acquisition. In chapter 5 the main findings made during this study and their impact on the literature are discussed.

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