Methods of gene transformation ï½‍ particle bombardment ï½‍

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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 Presentation

Transcript of Methods of gene transformation ï½‍ particle bombardment ï½‍

  • Methods of gene transformationparticle bombardment

  • IntroductionBiodiversity & Sexual reproductionReproductively isolated v.s gene transferBacterium, plants, animals, etc, have the same DNA

  • Methods of gene transformationForeign genes into the living cellBiological methodsAgrobacterium, Physical methodsParticle bombardmentProtoplastsElectroporationSilicon carbide whiskersMicroinjection , .etc.

  • Methods of gene transformationSilicon carbide whiskers

  • Methods of gene transformationmicro_pulser electroporator

  • Methods of gene transformationMicroinjection

  • Particle bombardmentSanford, J.C. and co-workers (1987)

  • Particle bombardmentExperiment Small tungsten particles(average diameter 4m)Initial velocity of Particle leaving is about 430 m/sTissue : intact epidermal cells of Allium cepa (onion)

  • Particle bombardmentResult90% of the cells in a 1 cm2 of A. cepa epidermal tissue typically contain microprojectiles following bombardment.

  • Particle bombardmentRelationship betwwen cell survival and the number of microprojectiles within a cell.

  • Particle bombardmentExperiment TMV(isolated from tobacco mosaic virus)Small tungsten particles(average diameter 4m)A. cepa cellsExamine the bombarded cells microscopically for the presence of viral inclusion bodies(crystallized virus particles) 3 days after treatment

  • Particle bombardmentResultIn the cytoplasm and vacuole of 30~40% of the cells that contained microprojectiles.

    Be comparable to electroporation etc.

  • Particle bombardmentExperiment Tungsten microprojectiles were coated with plasmid gene----p35S-CATA. cepa epidermal tissueCAT assay

  • Particle bombardmentResult

  • Particle bombardmentTo sum upMicroprojectiles 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 bombardmentPower sourcegunpower , He , N2 Microprojectilestungstem or gold This process has been called the microprojectile bombardment method, the gene gun method, the particle acceleration method, biolistic processetc.

  • Particle gun designs

  • Particle gun designs

  • Particle gun designs