Methods of gene transformation ～ particle bombardment ～

1. Methods of gene transformation～particle bombardment～ 簡維瑩 南師 自碩一

2. Introduction • Biodiversity & Sexual reproduction • Reproductively isolated v.s gene transfer • Bacterium, plants, animals, …etc, have the same 遺傳物質—DNA

3. Methods of gene transformation • Foreign genes into the living cell • Biological methods • Agrobacterium, … • Physical methods • Particle bombardment • Protoplasts • Electroporation • Silicon carbide whiskers • Microinjection , ….etc.

4. Methods of gene transformation Silicon carbide whiskers

5. Methods of gene transformation micro_pulser electroporator

6. Methods of gene transformation Microinjection

7. Particle bombardment • Sanford, J.C. and co-workers (1987)

9. Particle bombardment • Experiment 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)

10. Particle bombardment • Result： • 90% of the cells in a 1 cm2 of A. cepa epidermal tissue typically contain microprojectiles following bombardment.

11. Particle bombardment Relationship betwwen cell survival and the number of microprojectiles within a cell.

12. 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

13. Particle bombardment • ResultⅠ： • In the cytoplasm and vacuole of 30~40% of the cells that contained microprojectiles. • Be comparable to electroporation …etc.

14. Particle bombardment • Experiment Ⅲ • Tungsten microprojectiles were coated with plasmid gene----p35S-CAT • A. cepa epidermal tissue • CAT assay

15. Particle bombardment • ResultⅡ：

16. 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.

17. Particle bombardment • Power sourcegunpower , He , N2… • Microprojectilestungstem or gold • This process has been called • the microprojectile bombardment method, • the gene gun method, the particle • acceleration method, • biolistic process…etc.

18. Particle gun designs

19. Particle gun designs

20. Particle gun designs