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Gene Transfer System for Plant Seed, Egg and Microbe SONIDEL Limited

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

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Gene Transfer System for Plant Seed, Egg and Microbe SONIDEL Limited

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  1. 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.

  2. 1) Developmentof 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)

  3. 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)

  4. 2) Basic Technique and Feature Conventional Electroporation uses Electrical pulse only This new technique uses 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)

  5. 2) Basic Technique and Feature Fig.1: Vacuum Chamber (left)and Electroporator, CUY21EDIT(right) Fig.2: Chamber Having Platinum Electrode, CUY495P10 (5/24)

  6. 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)

  7. Transferred pWI vector integrated npt II gene 3) Transformed Rice Plant Cultivation (not transformed) (npt II gene introduced ) Fig2. Fertilizable transformed plant Fig.1Geneticine selection of rice seeds and/or seedlings 1 2 3 4 5 6 7 8 9 10 M 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)

  8. 3) Transformed Rice Plant Cultivation RT-PCR : T1nptⅡ gene detection (Rice Plant) (8/24)

  9. 4) Improvement of the method of gene transfer Fig2. Ultrasonic treatment Fig1. Hexagonal chamber type electrode, CUY602 (simple determination) Kiatake Koshihikari Score After improvement When we started our research Fig.3 Polarity exchange device, CUY901 Fig.4 Result of improvement ・When we started our research In waterVacuum EP Growing ・After improvement In waterUltrasonicVacuum Polarity exchangeEP Ultrasonic Growing (9/24)

  10. 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)

  11. 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)

  12. 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)

  13. Gene Transfer to Silk Worm Eggs ③ Gene Transfer Condition Setup：　 Pulse length 10ms, Pulse interval50ms, No. of Pulses 10 Optimized Voltage GFP Expression Rate Hatching rate ％ Voltage（V) Fig.1 Electrical condition, GFP expression rate and Hatching rate (13/24)

  14. 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)

  15. 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)

  16. Gene Transfer to Arabidopsis Seeds　② Gene Transfer Condition Setup Voltage：30Ｖ Pulse length：50ms Pulse interval：75ms GFP Expression Rate GFP Expression Rate (%) Number of Pulses Fig.1 GUS Gene Expression in Arabidopsis Seeds Transfered EL2Ω(35S+Ω+GUS) 99 Pulses leads to a good result (16/24) GFP and npt II gene are also transfered.

  17. 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)

  18. Gene Transfer to Yeast ① Gene Transfer Procedure Vector DNA Specific name Saccharomyces 　　　　 cerevisiae StrainBY4700 　　　　 (Sequenced all its genome) GenotypeMATa ura3Δ0 Ura3 / Orotidine-5‘-phosphate decarboxylase missing Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Yamaguchi University Host Yeast Amylase gene Unable to culture in urasil missing medium (minimum medium) Yeast that can not synthesize uracil Uracil synthesis gene Gene Transfer Select transferred gene in minimum medium Minimum medium recombinant Uracil synthesis gene Amylase gene Recombinant secretes mold amylase Amylolysis part by amylase Starch iodine reaction Starch medium Fig.1 Procedure for Gene Transfer to Yeast (18/24)

  19. Gene Transfer to Yeast ② Cultivation of Yeast Transfectant Wild type (19/24)

  20. Gene Transfer to Yeast ③ 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 V+EP is best (20/24)

  21. 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)

  22. Gene Transfer by Noncontact type Ultrasonic Processor ① Apparatus A.Appearance B.Inside of the tank (22/24)

  23. Gene Transfer by Noncontact type Supersonoc Processor ② 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)

  24. Gene Transfer by Noncontact type Ultrasonic Processor ③ Transfer Results CNT 　 　5min 　 　１０min B. Cultured rice cells : pWI-GUS A. Rice mature seeds CNT 　　5min 　 １０min (24/24) C.Cultured tobacco cells : EL2Ω

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