Methods of gene transformation ～ particle bombardment ～

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