Gene Transfer System

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Gene 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.
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1) 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)
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1) 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)
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2) 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)
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2) Basic Technique and Feature
Fig.1 Vacuum Chamber (left) and Electroporator,
CUY21EDIT(right)

Fig.2 Chamber Having Platinum Electrode,
CUY495P10
(5/24)
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2) 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)
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3) 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)
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3) 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)
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4) Improvement of the method of gene transfer

(9/24)
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5)New experiments in 2007 or later

• Gene transfer to Silk Worm Eggs

• Gene transfer to Arabidopsis seeds

• Gene transfer to Yeast

• Contact-free Ultrasonic Apparatus

(10/24)
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Gene 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)
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Gene Transfer to Silk Worm Eggs ? Our method
(Vacuum and Electroporation)
(12/24)
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Gene Transfer to Silk Worm Eggs ? Gene Transfer
Condition
Setup Pulse length 10ms, Pulse interval 50ms,
No. of Pulses 10
(13/24)
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5)New experiments in 2007 or later

• Gene transfer to Silk Worm Eggs

• Gene transfer to Arabidopsis seeds

• Gene transfer to Yeast

• Contact-free Ultrasonic Apparatus

(14/24)
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Gene Transfer to Arabidopsis Seeds ? Micro seeds,
Electrode for Microbe and Tube
(15/24)
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Gene Transfer to Arabidopsis Seeds ? Gene
Transfer Condition
(16/24)
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5)New experiments in 2007 or later

• Gene transfer to Silk Worm Eggs

• Gene transfer to Arabidopsis seeds

• Gene transfer to Yeast

• Contact-free Ultrasonic Apparatus

(17/24)
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Gene 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
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Gene Transfer to Yeast ? Cultivation of Yeast


(19/24)
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Gene 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)
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5)New experiments in 2007 or later

• Gene transfer to Silk Worm Eggs

• Gene transfer to Arabidopsis seeds

• Gene transfer to Yeast

• Noncontact type Ultrasonic Processor

(21/24)
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Gene Transfer by Noncontact type Ultrasonic
Processor ? Apparatus

A. Appearance
B. Inside of the tank

(22/24)
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Gene 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)
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Gene 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