Transcript Gene Transfer System for Plant Seed, Egg and Microbe

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.
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)
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)
2) Basic Technique and Feature
Conventional Electroporation
This new technique uses
uses Electrical pulse only
Vacuum + Electrical pulse
Basic Procedure (e.g. Rice Plant）
① Soak mature seeds in water overnight.
② Soak mature seeds in electroporation buffer
③ Strong Vacuum treatment→DNA permeate tissues ・・・Vacuum Chamber
（Fig.1）
④ Electroporation→DNA is transferred into cells
・・・Electroporator（Fig.1）, Chamber Having Platinum Electrode（Fig.2）
(4/24)
2) Basic Technique and Feature
Fig.1: Vacuum Chamber (left) and Electroporator,
CUY21EDIT(right)
Fig.2: Chamber Having Platinum Electrode, CUY495P10
(5/24)
2) Basic Technique and Feature
Effectiveness
500
400
300
200
100
VE
0
N
--
V -
E
--
V E
N: Negative control, --: Buffer only, V: Vacuum, E: Electroporation
Fig.3: The level of GUS gene expression by
fluorescence assay (Wheat)
E
Fig. 4: GUS gene expression
reference (Wheat)
(6/24)
3) Transformed Rice Plant Cultivation
Transferred pWI vector integrated npt II gene
(not transformed)
(npt II gene introduced )
Fig.1 Geneticine selection of rice seeds and/or seedlings
Fig2. Fertilizable transformed plant
M wt
M
1 2 3 4 5 6 7 8 9 10 11 wt
9.4-
1 2 3 4 5
1 2 3 4 5 6 7 8 9 10
6 7 8
9.4-
-5 cp
-1 cp
4.4-
4.4-
-2 cp
-10 cp
Fig3. Southern analysis in
T0(existent) generation
Fig4. Next generation (T1)
individual
Fig5. . Southern analysis of
T1(next) generation
(7/24)
3) Transformed Rice Plant Cultivation
RT-PCR : T1 nptⅡ gene detection (Rice Plant)
No. of T0
T1 of T1
PCR+No.
RT-PCR+No.
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)
4) Improvement of the method of gene transfer
Fig2. Ultrasonic treatment
Fig1. Hexagonal chamber type
electrode, CUY602
(simple determination)
Koshi
hikari
Score
Kiat
ake
Fig.3 Polarity exchange device,
CUY901
・When we started our research
In water
Vacuum
When we started
our research
After
improvement
Fig.4 Result of improvement
EP
Growing
・After improvement
In water
Ultrasonic
Vacuum
Polarity exchange EP
Ultrasonic
Growing
(9/24)
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)
Gene Transfer to Silk Worm Eggs ①
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)
Gene Transfer to Silk Worm Eggs ②
Our method (Vacuum and Electroporation)
A. 2 days after gene transfer
B. 10 days after gene transfer
The capacity is
approx. 2,000 eggs
at one time
Make mass transfer possible
C. Hatchling Larva
(12/24)
Gene Transfer to Silk Worm Eggs ③
Gene Transfer Condition
Optimized
Voltage
100
GFP発現割合
Hatching rate
ふ化率
GFP Expression Rate
80
％
Setup：
Pulse length 10ms,
Pulse interval 50ms,
No. of Pulses 10
60
40
20
0
10 25
50 75 100 125 150 200
Voltage（V)
Fig.1 Electrical condition, GFP expression rate and Hatching rate
(13/24)
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)
Gene Transfer to Arabidopsis Seeds ①
Micro seeds, Electrode for Microbe and Tube
Fig.2 GUS gene expression
in tobacco BY-2 cells
Fig.1 Platinum Plate Electrode for 2ml Tube,
CUY280
Fig.3 GUS gene expression
in Arabidopsis Seeds
(15/24)
Gene Transfer to Arabidopsis Seeds ②
Gene Transfer Condition
Setup
Voltage：30Ｖ
Pulse length：50ms
Pulse interval：75ms
7
Rate (%)
GFP Expression
G US発現割合(%)
6
GFP Expression Rate
GUS発現割合
5
4
3
2
1
0
10
20
50
99
パルス回数
Number
of Pulses
Fig.1 GUS Gene Expression in Arabidopsis Seeds
Transfered EL2Ω(35S+Ω+GUS)
99 Pulses leads to a good result
GFP and npt II gene are also transfered.
(16/24)
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)
Gene Transfer to Yeast ①
Gene Transfer Procedure
Vector DNA
Host Yeast
Amylase
gene
Yeast that can not
synthesize uracil
Uracil
synthesis
gene
Specific name Saccharomyces
Unable to culture in
urasil missing medium
(minimum medium)
Gene Transfer
Select transferred gene in minimum medium
cerevisiae
Strain BY4700
(Sequenced all its genome)
Genotype MATa ura3Δ0
Ura3 / Orotidine-5‘-phosphate
decarboxylase missing
Minimum medium
recombinant
Uracil
synthesis
gene
Amylase
gene
Department of Applied Chemistry and Chemical
Engineering,
Faculty of Engineering,
Yamaguchi University
Recombinant secretes mold amylase
Amylolysis part
by amylase
Starch iodine
reaction
Starch medium
Fig.1 Procedure for Gene Transfer to Yeast
(18/24)
Gene Transfer to Yeast ②
Cultivation of Yeast
Transfectant
Wild type
(19/24)
Gene Transfer to Yeast ③
Efficiency of Yeast Transformation
A. Heat Shock Method
（conventionally）
コロニー数
200
150
100
No. of
コロニー数
Colony
50
0
HS
V
EP
V+EP
Fig.1 Efficiency of Yeast Transformation
HS : Heat Shock
V: Vacuum
EP : Electroporation
V + EP : Vacuum + Electroporation
B. Vacuum +
Electroporation
V+EP is best
(20/24)
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)
Gene Transfer by Noncontact type Ultrasonic Processor ①
Apparatus
A. Appearance
B. Inside of the tank
(22/24)
Gene Transfer by Noncontact type Supersonoc Processor ②
Structure
Attachment for fixing
Tube or vial in which biologic
material (e.g. seeds) are
contained
Tempraturecontrolled
water tank
Supersonic
probe
Supersonic from 3 ways (left, right, bottom) crossing in the
material position gives maximum energy.
Supersonic
probe
(23/24)
Gene Transfer by Noncontact type Ultrasonic Processor ③
Transfer Results
CNT
A. Rice mature seeds
CNT
5min
5min
１０min
B. Cultured rice cells : pWI-GUS
１０min
C. Cultured tobacco cells : EL2Ω
(24/24)