Title: Gene Transfer System
1Gene Transfer System for Plant Seed, Egg and
Microbe SONIDEL Limited
Resarch cooperation National Institute of
Agrobiological Sciences Plant Genetic Engineering
Unit
Improvement Research is being Made.
21) Development of Concept
Current Gene Transfer Method
Rice Agrobacterium Wheat, Barley Particle
Gun
Need Tissue Culture (time-consuming !)
Silk Worm Microinjection
Need High Skill A Lot of Labor (tedious !)
(2/24)
31) Development of Concept
A New Gene Transfer Method
National Institute of Agrobiological Sciences
Japan patents pending
Plant No Need Tissue Culture Animal (silk
worm) No Need Microinjection
- Simple
- Efficient
- Low-cost
Electroporation method including the use of
depressurization / presurization PCT/JP03/08937
(3/24)
42) Basic Technique and Feature
This new technique uses Vacuum Electrical pulse
Conventional Electroporation uses Electrical
pulse only
Basic Procedure (e.g. Rice Plant)
? Soak mature seeds in water overnight. ? Soak
mature seeds in electroporation buffer ? Strong
Vacuum treatment?DNA permeate tissues Vacuum
Chamber(Fig.1) ? Electroporation?DNA is
transferred into cells Electroporator(Fig.1),
Chamber Having Platinum Electrode(Fig.2)
(4/24)
52) Basic Technique and Feature
Fig.1 Vacuum Chamber (left) and Electroporator,
CUY21EDIT(right)
Fig.2 Chamber Having Platinum Electrode,
CUY495P10
(5/24)
62) Basic Technique and Feature
Effectiveness
VE
E
--
N Negative control, -- Buffer only, V
Vacuum, E Electroporation
Fig. 4 GUS gene expression reference (Wheat)
Fig.3 The level of GUS gene expression by
fluorescence assay (Wheat)
(6/24)
73) Transformed Rice Plant Cultivation
Transferred pWI vector integrated npt II gene
(not transformed) (npt II gene introduced )
Fig2. Fertilizable transformed plant
Fig.1 Geneticine selection of rice seeds and/or
seedlings
M
1 2 3 4 5 6 7 8 9 10
wt
M
wt
1 2 3 4 5 6 7 8 9 10 11
1 2 3 4 5 6 7 8
9.4-
9.4-
-5 cp
-1 cp
4.4-
4.4-
-2 cp
-10 cp
Fig5. . Southern analysis of T1(next) generation
Fig3. Southern analysis in T0(existent) generation
Fig4. Next generation (T1) individual
(7/24)
83) Transformed Rice Plant Cultivation
RT-PCR T1 npt? gene detection (Rice Plant)
No. of T0 T1 of T1 PCRNo. RT-PCRNo.
1 7 6 6
2 15 10 9
3 9 9 9
4 8 8 8
5 12 11 11
6 9 5 5
7 18 17 16
8 6 5 5
(ongoing)
(8/24)
94) Improvement of the method of gene transfer
(9/24)
105)New experiments in 2007 or later
- Gene transfer to Silk Worm Eggs
- Gene transfer to Arabidopsis seeds
- Contact-free Ultrasonic Apparatus
(10/24)
11Gene Transfer to Silk Worm Eggs ? Current common
method
Inject Vector and Plasmid into an egg
Breeding Mounting Mating
Extraction
Feeding Artificial diet
Screen by fluorescence stereomicroscope
After making a hole by a thin needle, inject DNA
by glass capillary
A lot of care!
(11/24)
12Gene Transfer to Silk Worm Eggs ? Our method
(Vacuum and Electroporation)
(12/24)
13Gene Transfer to Silk Worm Eggs ? Gene Transfer
Condition
Setup Pulse length 10ms, Pulse interval 50ms,
No. of Pulses 10
(13/24)
145)New experiments in 2007 or later
- Gene transfer to Silk Worm Eggs
- Gene transfer to Arabidopsis seeds
- Contact-free Ultrasonic Apparatus
(14/24)
15Gene Transfer to Arabidopsis Seeds ? Micro seeds,
Electrode for Microbe and Tube
(15/24)
16Gene Transfer to Arabidopsis Seeds ? Gene
Transfer Condition
(16/24)
175)New experiments in 2007 or later
- Gene transfer to Silk Worm Eggs
- Gene transfer to Arabidopsis seeds
- Contact-free Ultrasonic Apparatus
(17/24)
18Gene Transfer to Yeast ? Gene Transfer Procedure
Specific name Saccharomyces
cerevisiae Strain BY4700 (Sequenced all
its genome) Genotype MATa ura3?0 Ura3 /
Orotidine-5-phosphate decarboxylase
missing Department of Applied Chemistry and
Chemical Engineering, Faculty of
Engineering, Yamaguchi University
(18/24)
Fig.1 Procedure for Gene Transfer to Yeast
19Gene Transfer to Yeast ? Cultivation of Yeast
(19/24)
20Gene Transfer to Yeast ? Efficiency of Yeast
Transformation
A. Heat Shock Method (conventionally)
No. of Colony
Fig.1 Efficiency of Yeast Transformation
B. Vacuum Electroporation
HS Heat Shock V Vacuum EP Electroporation
V EP Vacuum Electroporation
VEP is best
(20/24)
215)New experiments in 2007 or later
- Gene transfer to Silk Worm Eggs
- Gene transfer to Arabidopsis seeds
- Noncontact type Ultrasonic Processor
(21/24)
22Gene Transfer by Noncontact type Ultrasonic
Processor ? Apparatus
A. Appearance
B. Inside of the tank
(22/24)
23Gene Transfer by Noncontact type Supersonoc
Processor ? Structure
Tube or vial in which biologic material (e.g.
seeds) are contained
Attachment for fixing
Temprature-controlled water tank
Supersonic probe
Supersonic probe
Supersonic from 3 ways (left, right, bottom)
crossing in the material position gives maximum
energy.
(23/24)
24Gene Transfer by Noncontact type Ultrasonic
Processor ? Transfer Results
CNT 5min 10min
B. Cultured rice cells pWI-GUS
A. Rice mature seeds
CNT 5min 10min
(24/24)
C. Cultured tobacco cells EL2O