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Methods of gene transformation ~particle bombardment~ 簡維瑩 南師 自碩一 Introduction Biodiversity Sexual reproduction Reproductively isolated v.s gene transfer Bacterium, plants, animals, …etc, have the same 遺傳物質—DNA & Methods of gene transformation Foreign genes into the living cell Biological methods Agrobacterium, Physical Particle … methods bombardment Protoplasts Electroporation Silicon carbide whiskers Microinjection , ….etc. Methods of gene transformation Silicon carbide whiskers Methods of gene transformation micro_pulser electroporator Methods of gene transformation Microinjection Particle bombardment Sanford, J.C. and co-workers (1987) Particle bombardment Experiment Small Ⅰ tungsten particles(average diameter 4µm) Initial velocity of Particle leaving is about 430 m/s Tissue : intact epidermal cells of Allium cepa (onion) Particle bombardment Result: of the cells in a 1 cm2 of A. cepa epidermal tissue typically contain microprojectiles following bombardment. 90% Particle bombardment Relationship betwwen cell survival and the number of microprojectiles within a cell. Particle bombardment Experiment Ⅱ TMV(isolated from tobacco mosaic virus) Small tungsten particles(average diameter 4µm) A. cepa cells Examine the bombarded cells microscopically for the presence of viral inclusion bodies(crystallized virus particles) 3 days after treatment Particle bombardment ResultⅠ: In the cytoplasm and vacuole of 30~40% of the cells that contained microprojectiles. Be comparable to electroporation …etc. Particle bombardment Experiment Tungsten Ⅲ microprojectiles were coated with plasmid gene----p35S-CAT A. cepa epidermal tissue CAT assay Particle bombardment ResultⅡ: Particle bombardment To sum up… Microprojectiles pierce cell walls and membranes and enter intact plant cells without killing them. Microprojectiles were used to carry RNA or DNA into epidermal tissue of onion and these molecules were subsequently expressed genetically. It would provide a broadly applicable transformation mechanism. Particle bombardment sourcegunpower , He , N2 … Microprojectilestungstem or gold Power This process has been called the microprojectile bombardment method, the gene gun method, the particle acceleration method, biolistic process…etc. Particle gun designs Particle gun designs Particle gun designs