Establish a novel stress-detecting system by using Annexin V-marked apoptotic cell in zebrafish (Danio rerio)

• Main Authors: Shih-Feng Pan, 潘世鋒
• Other Authors: Guor-Mour Her
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/16600435082655320559

碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 101 === Multicellular organisms to eliminate unneeded and damaged cells in a process by apoptosis. When the organism to external stimuli, such as UV irradiation or drug-induced, resulting in cell DNA damage. Organism will initiate apoptosis to clear damaged cells. In addition, apoptosis during embryonic development are also involved in the pattern of individual organisms sculpture, and new cells and apoptotic cells must be in equilibrium in the organism. Many diseases have been linked to new cells and apoptotic cells state imbalances, such as neurodegenerative diseases, immunedeficiency, autoimmune reactions and cancer. Therefore, the study of apoptosis is more and more attention. Commonly used in vitro apoptosis detection methods such as TUNEL or utilize nucleic acid dye acridine orange, while the detection of apoptosis in live animals is less to be raised.Therefore, we have designed a full name apoptosis cell death(ACD) gene sequences to detect apoptosis in vivo. The sequences is drived by β-actin promoter, connect with Nitroreductase (NTR) gene behind promoter.Using Nitroreductase (NTR) can convert Metronidazole (Mtz) into cytotoxic to promote apoptosis. The middle splited by 2A3, and connect with secA5 behind 2A3. Using Annexin V binds phospholipids with high affinity, as the indicator of apoptosis. We microinjected this sequence into zebrafish embryos. And screening with this gene of fish (F0),which mating with PL zebrafish to produce progeny (F1). Then by F1 self-cross to produce with homozygous F2, and using F2 for subsequent experiments. Using can promote apoptosis drugs such as camptothecine,rotenone,colchicine,amitriptyline and metronidazole for dosing tretment,or impose a UV irradiation and heat shock.Fluorescence was observed after treatment performance can be seen as punctate green fluorescent labeled apoptotic cells. Thus, the use of zebrafish performance ACD sequence can be used as a model organism,facilitate the subsequent apoptosis of related reserch.