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Developing a Zebrafish Model System for Thrombocyte Research

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Developing a Zebrafish Model System for Thrombocyte Research Developing a zebrafish model system for thrombocyte research Master Degree Project in Systems Biology A2E, SY749A Two years Level ECTS Spring and Autumn 2017 (2018-01-19) Author: Aida Kaffash Hoshiar [email protected] Molecular Biotechnology (systems biology) Supervisor: Marcel den Hoed [email protected] Dept. of Immunology, Genetics and Pathology, Uppsala University Co-supervisor: Ci Song [email protected] Dept. of Medical Sciences, Uppsala University Examiner: Diana Tilevik [email protected] School of Bioscience, University of Skövde Högskolan i Skövde Högskolevägen Box 408, 541 28 Skövde Sweden Abstract Platelets are blood cells produced from megakaryocytes, and their function is to stop bleeding in injury time. Low platelet count cause disorders like thrombocytopenia and abnormal bleeding. Thanks to 70% similarity of zebrafish genes to human, developing a transgenic zebrafish model system with fluorescently labeled thrombocytes and red blood cells can help us study drugs effects on platelets. Genome-wide association studies have identified several loci linked to platelet count in humans. This study aimed to develop and validate a zebrafish model system to study thrombocyte related research using aspirin as a drug well characterized effects. Then, generate multiplex mutant zebrafish line for thrombocyte count related genes. By taking advantage of CRISPR-Cas9 genome- editing technology, a mutant zebrafish model was generated to study seven candidate genes related to platelet count in human. To examine the effect of aspirin, larvae at four days post fertilization were treated with two doses of aspirin (5 µg/ml and 45 µg/ml) for 24 h. At five day post fertilization treated and control groups were imaged for thrombocyte count and body size using a high- throughput vertebrate automated screening technology BioImager. A mixed effect model was used in statistical analysis. The mean thrombocyte count in the control group (n=152), as well as in the group treated with 5 µg/ml (n=150) and 45 µg/ml (n=154) aspirin was 14.65 ± 4.24, 15.54 ± 4.11, and 15.65 ± 4.77 respectively. The ranges of mean thrombocyte count in all groups were 4.95 to 42.4. Only higher dose of aspirin (45 µg/ml) showed statistically significant increased on thrombocyte count (P<0.05). However, the body size factors were increased in both aspirin treated groups compared with controls (P<0.05). This may suggest an unknown role of aspirin in development stage that needs further investigation. The transgenic zebrafish model for thrombocyte and red blood cell could be used as a model system in thrombocyte related studies as well as drug screening. Popular scientific summary Blood is made of different kind of blood cells that are all produced from bone marrow. One type of these important cells is called platelets (in mammalian) or thrombocytes (in fish). If an injury occurs and causes bleeding, platelets aggregate together in order to stop the bleeding. When there is lack of platelet in blood, they cannot make clots to ban the bleeding and one can die from bleeding. Thrombocytopenia is a bleeding genetic disorder in low level of platelets. The treatment addresses the cause of disease that may refer to bone marrow or different unknown genetics variants. Genome-wide association studies have identified genetic variants associated with platelet count. A good way to examine which of the genes near these variants are the causal genes is to knock out the genes in an animal model system to see the effect on platelet count. Transgenic zebrafish larva with fluorescently labeled thrombocytes and red blood cells are a good model system to examine the function of the genes on blood cells. In current study, seven genes related to platelet count in human were knocked out in transgenic zebrafish using CRISPR-Cas9 technology (cas9 protein cuts specific part of double stranded DNA and cause insertion or deletion mutation in DNA sequence). Then, one can study the effect of those genes on blood cells count using high throughput screening under novel microscopy techniques. Aspirin is a well-known antiplatelet drug that is also widely used for other purposes, like to control fever, as a painkiller, and after heart attack. Long-term low doses of aspirin inhibit the aggregation of platelets by blocking formation of thromboxane A2 in humans. Thromboxane A2 is a protein that motivates the activity of other platelets and enhances platelets aggregation. Although people have been using aspirin for more than a century, its side effects, especially on development, are still incompletely understood. In this experiment, transgenic zebrafish larvae at four day post fertilization (dpf) were treated with two doses of aspirin (5 µg/ml and 45 µg/ml) for 24 h to see its effect on thrombocyte count as well as on body size. The low dose of aspirin (5 µg/ml) was reported to prevent thrombosis in 5 dpf zebrafish larvae. The selected high dose of aspirin (45 µg/ml) is 10% of the lethal concentration for 3-5 dpf larvae. The treated and control groups were imaged in 5 dpf for thrombocyte count and body size, followed by a statistical analysis to study the effect of aspirin on both thrombocyte count and body size. It was observed that 45 µg/ml resulted in a higher thrombocyte count in zebrafish larvae. In addition, because the larvae were in developing stage while treated with aspirin, body size was higher in both groups treated with low and high doses of aspirin compared with untreated controls. This may indicate an unknown molecular mechanism of aspirin’s target during the development process. However, in adult humans low dose of aspirin intake is 1.5 mg/kg (75 mg for 50 kg adult human) while in this study low dose of aspirin was 10,000 mg/kg (5 µg/ml for 0.5 mg larvae) and high dose of aspirin was 90,000 mg/kg (45 µg/ml for 0.5 mg larvae). An animal model system to study platelet related research, could increase our understanding on different molecular mechanism of drugs and their side effect in various doses. List of abbreviations CAD Coronary Artery Disease CRISPR Clustered Regularly Interspaced Short Palindromic Repeats dpf days post-fertilization DsRed Discosoma Red fluorescent protein eGFP enhanced Green Fluorescent Protein FLA Fragment Length Analysis GWAS Genome-wide association studies hpf hour post-fertilization HSCs Hematopoietic Stem Cells KLF1 Kruppel-Like Factor 1 MAP1A Microtubule Associated Protein 1A NFE2 Nuclear Factor Erythroid 2 PLT Platelet Count RBCs Red Blood Cells sgRNA single guide Ribonucleic Acid VAST Vertebrate Automated Screening Technology Table of Contents 1. Introduction ................................................................................................................................... 1 1.1. Platelet function and development ......................................................................................... 1 1.2. Zebrafish as a model to study blood cells ................................................................................ 2 1.3. Aspirin's antiplatelet effect ..................................................................................................... 2 1.4. Genome–wide association studies for PLT............................................................................... 3 1.5. CRISPR/cas9 technology .......................................................................................................... 4 1.6. Aims ....................................................................................................................................... 4 2. Material and methods ................................................................................................................... 4 2.1. Zebrafish handling, husbandry, and sorting transgenic zebrafish ............................................. 5 2.2. Aspirin treatment.................................................................................................................... 5 2.3. High throughput imaging on thrombocyte count and RBCs by VAST BioImager ....................... 5 2.4. Quantification of thrombocyte count ...................................................................................... 6 2.5. Body size analysis.................................................................................................................... 7 2.6. Statistical analysis ................................................................................................................... 7 2.7. Generation of transgenic zebrafish mutant lines with platelet related genes ........................... 8 2.7.1. CRISPR-Cas9 target design .................................................................................................... 8 2.7.2. Preparation of sgRNA oligos and FLA primers for ordering ................................................... 8 2.7.3. sgRNA preparation ............................................................................................................... 9 2.7.4. Microinjection...................................................................................................................... 9 3. Results ........................................................................................................................................... 9 3.1. Script annotation results for thrombocyte count ..................................................................... 9 3.2. Thrombocyte count and body size ........................................................................................ 10 3.3. The effect of aspirin
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