methods of gene transformation ～ particle bombardment ～
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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. - PowerPoint PPT PresentationTRANSCRIPT
Methods of gene transformation～ particle bombardment～
簡維瑩南師 自碩一
IntroductionBiodiversity & Sexual reproductionReproductively isolated v.s gene transferBacterium, plants, animals, …etc, have
the same 遺傳物質— DNA
Methods of gene transformationForeign genes into the living cellBiological methods
Agrobacterium, …Physical methods
Particle 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 bombardmentSanford, J.C. and co-workers (1987)
Particle bombardmentExperiment Ⅰ
Small tungsten particles(average diameter 4µm)
Initial velocity of Particle leaving is about 430 m/s
Tissue : intact epidermal cells of Allium cepa (onion)
Particle bombardmentResult ：
90% of the cells in a 1 cm2 of A. cepa epidermal tissue typically contain microprojectiles following bombardment.
Particle bombardment
Relationship betwwen cell survival and the number of microprojectiles within a cell.
Particle bombardmentExperiment Ⅱ
TMV(isolated from tobacco mosaic virus) Small tungsten particles(average diameter 4µm)
A. cepa cells Examine the bombarded cells microscopic
ally for the presence of viral inclusion bodies(crystallized virus particles) 3 days after treatment
Particle bombardmentResultⅠ ：
In the cytoplasm and vacuole of 30~40% of the cells that contained microprojectiles.
Be comparable to electroporation …etc.
Particle bombardmentExperiment Ⅲ
Tungsten microprojectiles were coated with plasmid gene----p35S-CAT
A. cepa epidermal tissue CAT assay
Particle bombardmentResultⅡ：
Particle bombardmentTo 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
Power sourcegunpower , He , N2 …
Microprojectilestungstem or gold
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