Transcript Methods of gene transformation ∼particle bombardment∼

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
 Foreign
genes into the living cell
 Biological methods
 Agrobacterium,
 Physical
 Particle
…
methods
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 bombardment
 Sanford,
J.C. and co-workers (1987)
Particle bombardment
 Experiment
 Small
Ⅰ
tungsten particles(average diameter 4µm)
 Initial velocity of Particle leaving is about
430 m/s
 Tissue : intact epidermal cells of Allium
cepa (onion)
Particle bombardment
 Result：
of the cells in a 1 cm2 of A. cepa
epidermal tissue typically contain
microprojectiles following bombardment.
 90%
Particle bombardment
Relationship betwwen cell survival and the number of
microprojectiles within a cell.
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
Particle bombardment
 ResultⅠ：
 In
the cytoplasm and vacuole of 30~40% of
the cells that contained microprojectiles.
 Be
comparable to electroporation …etc.
Particle bombardment
 Experiment
 Tungsten
Ⅲ
microprojectiles were coated with
plasmid gene----p35S-CAT
 A. cepa epidermal tissue
 CAT assay
Particle bombardment
 ResultⅡ：
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.
Particle bombardment
sourcegunpower , He , N2 …
 Microprojectilestungstem or gold
 Power
 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