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 4 µ m)  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 cm 2 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 4 µ m)  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  Result Ⅰ：  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  Result Ⅱ：

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, N 2 …  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