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Applications of Biotechnology in Agriculture for Water Security, Nguyen Van Thuan

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This presentation is part of the ProSPER.Net Young Researchers' School 2017 ‘Water Security for Sustainable Development in a Changing Climate’.

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Applications of Biotechnology in Agriculture for Water Security, Nguyen Van Thuan

  1. 1. Water Security for Sustainable Development in a Changing Climate Applications of Biotechnology in Agriculture For Water Security Nguyen Van Thuan, Ph.D Laboratory for cellular Reprogramming School of Biotechnology - International University
  2. 2. Human Bio-Organs Stem Cell Therapy Highest Quality Products for Agriculture Genome Preservation Assisted Reproductive Technology (ART) Preimplantation genetic diagnosis (PGD) Bio-medicine Research areas of Cellular Reprogramming Laboratory Lab for Genomic Reprogramming
  3. 3. Water Security for Sustainable Development in a Changing Climate Applications of Biotechnology in Agriculture For Water Security Nguyen Van Thuan, Ph.D Laboratory for cellular Reprogramming School of Biotechnology - International University
  4. 4. Why do we need the responsibility to protect WATER on our earth Freshwater Use Rivers, lakes & groundwater 0.25% of earth’s water 70% 20% 8% Industry The earth’s WATER 0.01% of earth’s water Useable Earth’s water useable is very small and not as infinite as we thought
  5. 5. Why do we need the responsibility to protect WATER on our earth Drought Flood We'll need to use water much more smartly
  6. 6. Why do we need the responsibility to protect WATER on our earth Loss of 15-20 % of body water we will death SMART Water use is very important Without water for 1 week, human will be death Water in human body
  7. 7. SMART Water use in Agriculture In industry: SMART ENERGY In Agriculture: SMART Horticulture SMART Livestock Farming??
  8. 8. In Agriculture: Livestock Farming System SMART Livestock Farming?? SMART Water use in Agriculture
  9. 9. SMART Water Use in Livestock Farming Traditional livestock farming Smart livestock farming Applications of high- biotechnology to the farm
  10. 10. 1. Advances in embryos technology: intracytoplasmic sperm injection (ICSI) Advanced Biotechnologies in Animals 2. Advances in cloning animals: Applications in Agriculture 3. Advances in transgenic animals Applications in Agriculture & in Medicine 4. Advances in animal biotechnology in regenerative biomedicine: Pharming
  11. 11. Nguyen Van Thuan, Ph.D. Department of Bioscience & Biotechnology Konkuk University, Seoul, Korea vanthuan@konkuk.ac.kr Advance Embryo Technology
  12. 12. Fertilization Sperm Egg (oocyte) Zygote 8-cell embryo The first differentiation Blastocyst Preimplantation development What is the EMBRYO?
  13. 13. What is Embryo Technology? Embryo Technology is technology based on Embryology Embryological sciences: • Fertilization • Characteristic of embryos development • Zygotic gens activation • Epigenetic reprogramming during embryo development • Differentiation of embryos To study Embryo Technology
  14. 14. Fertilization Sperm Egg (oocyte) Fertilization: beginning of new organism
  15. 15. Infertile Treatment: ICSI
  16. 16. Embryos transfer (ET) Embryos with high quality Supper dairy cow ET
  17. 17. Preservation for Mouse Spermatozoa Without Freezing vanthuan@konkuk.ac.kr Nguyen Van Thuan, Ph.D Department of Animal Biotechnology Konkuk University Van thuan et al. BOR 2005
  18. 18. Development of normal mice from oocytes injected with freeze-dried spermatozoa Instant sperm: Just add water Wakayama T, and Yanagimachi R. 1998.
  19. 19. Live/dead cell staining Fresh sperm After freeze-dried Freeze-dried sperm were all died Wakayama T, and Yanagimachi R. 1998.
  20. 20. However… Freeze-dried sperm can be activated oocytes and supported full-term development after ICSI. 40 30 20 10 0 1 day 1 week 1 month 3 months % 200 C (Room temperature) 40 C Wakayama et al. Nat Bio 1998. 世界初!フリーズドライ精子から生まれたマウス Dry-mon
  21. 21. For that reasons In order to make freeze-dried sperm, we still need liquid nitrogen, freeze-dry system…. However, Offspring derived from dead sperm by ICSI BIOLOGY OF REPRODUCTION, 2005
  22. 22. Applications of ICSI - Sperm preservation - Embryo preservation - Embryo transfer - Transgenic Animals - Treatment of infertile diseases - etc… Assisted Reproductive Techniques (ART)
  23. 23. 24 Rescues VN Bos Garus (bò tót Việt Nam) Bos gaurus dead on May 26, 2014 at Quang Nam May 30, 2014 Tinh trùng bò tót (Spermatozoa of Bos gaurus)Can activation oocytes
  24. 24. Water Security for Sustainable Development in a Changing Climate Advances in somatic cell nuclear transfer (SCNT): Cloning animals Nguyen Van Thuan, Ph.D Laboratory for cellular Reprogramming School of Biotechnology - International University
  25. 25. Somatic Cell Nuclear Transfer: Cloned Animals Skin cells (Differentiated cells) Reprogramming Stem cells (Undifferentiated cells) Dolly, the word’s first cloned animal, 1996 1997 1998 2000 2000 2002 2002 2003 2003 2005 2009 Center for Developmental Biology Japan Dr. Wakayama Dr. Kishigami Genomic Reprogramming Lab. 2002 Nature Protocols, 2006 Production of cloned mice by somatic cell nuclear transfer Satoshi Kishigami, Sayaka Wakayama, Van Thuan Nguyen, Hiroshi Ohta, Eiji Mizutani, Takafusa Hikichi, Hong-Thuy Bui, Sebastian Balbach, Atsuo Ogura, Michele Boiani and Teruhiko Wakayama. Histone deacetylase inhibitor scriptaid rescues full-term development in cloned inbred mice by enhancing nascent mRNA production. Reproduction 2009 Van Thuan Nguyen , Hong-Thuy Bui, Takafusa Hikichi, Sayaka Wakayama, Satoshi Kishigami, Eiji Mizutani and Teruhiko Wakayama.
  26. 26. Somatic cell nucleus donor Oocyte donor Oocyte collection Donor cells collection Enucleation Remove donor cell cytoplasm Activation with CB for 6 hrs Embryo culture Surrogate mother Embryo transfer Cloned mice Caesarian section at 19.5dpc Nuclear transfer Subsequently culture for 1-2 hr ntES cells Bl Embryo culture Success rate is very low 1-2% Somatic Cell Nuclear Transfer: Cloned Animals
  27. 27. Somatic Cell Nuclear Transfer: Cloned Animals
  28. 28. The cytoplasm of mouse germinal vesicle (GV) stage oocytes can enhance somatic cell nuclear reprogramming Development. 2008 Bui HT, Wakayama S, Kishigami S, Kim JH, Van Thuan N Mouse GV oocytes extract Mouse fibroblast cells Treatment Mouse fibroblast cells Somatic cell nuclear transfer Mouse GV extract treatment improve the full-term development of cloned mouse Oct4 2 3 4SC ES DMEM medium 1 2 3 4 ES medium 1 LMNA GAPDH Weeks GV extracts induce expression of Oct-4, and repression of LMNA Mouse fibroblast cells Mouse GV oocytes extract Treatment Somatic Cell Nuclear Transfer: Cloned Animals
  29. 29. Epigenetic reprogramming in somatic cells induced by extract from germinal vesicle stage pig oocytes Development. 2012 Hong-Thuy Bui, D-N Kwon, M-H Kang, M-H Oh, M-R Park, W-J Park, S-S Paik, N. Van Thuan Pluripotent stem cells Pig GV oocytes extract In vivo redifferentiate into three germ layers Pig fibroblast cells Somatic Cell Nuclear Transfer: Cloned Animals
  30. 30. 25 generation Cloned generation 1 Cloned generation 2 We are the first time can increase the success rate of cloned animals from 1% to 10% Tế bào cơ thể 2012 However, the success rate of cloned mouse still low Histone deacetylase inhibitor scriptaid rescues full-term development in cloned inbred mice by enhancing nascent mRNA production. Reproduction 2009 Van Thuan Nguyen , Hong-Thuy Bui, Takafusa Hikichi, Sayaka Wakayama, Satoshi Kishigami, Eiji Mizutani and Teruhiko Wakayama. Somatic Cell Nuclear Transfer: Cloned Animals
  31. 31. The first time on the world we have successes 25 generations cloned mice from a body cell. Sayaka Wakayama, Takashi Kohda, Haruko Obokata, Mikiko Tokoro, Yukari Terashita, Eiji Mizutani, Nguyen Van Thuan, Satoshi Kishigami, Fumitoshi Ishino, and Teruhiko Wakayama Successful Serial Recloning in the Mouse over Multiple Generations CELL (Cell Stem Cell) 2013 Somatic Cell Nuclear Transfer: Cloned Animals
  32. 32. Conservation of Infertile and Rare Animals
  33. 33. 2.5 years old GFP-ICR male Our unpublished data Cloned GFP-ICR Rescue infertile age male mouse via Cloning Technique
  34. 34. Cloned female mice from donor cells of 3 year old ICR female by scriptaid treatment Our unpublished data
  35. 35. Next generations from male and female infertile Rescue infertile age female mouse via Cloning Technique Our unpublished data
  36. 36. Rescue Rare Animals (Bos gaurus) Somatic cell nuclear transferBos gaurus dead on May 26, 2014 at Quang Nam Bos gaurus cloned blastocyst This research is funded by Vietnam National University HoChiMinh City (VNU-HCM) under grant number B2016-28-01.
  37. 37. Application of Cloning Technique Production of healthy cloned mice from bodies frozen at -20 degrees C for 16 years. Wakayama et al, PNAS 2008
  38. 38. Advances in Somatic cell Reprogramming 1. Induced pluripotent stem cells: iPS cells OCT4 Sox2 c-Myc Klf4 Mouse fibroblast cells Kazutoshi Takahashi & Shinya Yamanaka Cell, 2006 Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors
  39. 39. Differentiation and transplantation of functional pancreatic beta cells generated from induced pluripotent stem cells derived from a type 1 diabetes mouse model. Stem Cells and Development. 2012 Jeon K, Lim H, Van Thuan N et al., OCT4 Sox2 c-Myc Klf4 Diabestes Mouse fibroblast cells iPS Cells Transplantation into diabetes mouse Decreases glucose levels Differentiation into pancreatic cells Pancreatic cells Advances in Somatic cell Reprogramming
  40. 40. Offspring derived from iPS cell Reprogramming by iPS cell iPS cells will inject into tetraploid embryos. Can we get offspring derived from iPS cells??? ??
  41. 41. Tetraploid blastocyst (4n) Injection ES cells Fetus was developed from ES cells Placenta was developed from tetraploid embryo Chromosome remodeling and differentiation of tetraploid embryos during preimplantation development. Part MR, Lee AR, Bui HT, Park KK, Cho SG, Song H, Van Thuan N Developmental Dynamics, 2011. Offspring derived from iPS cell
  42. 42. Somatic Cell Reprogramming: Applications in high quality of meat & Regenerative Biomedicine
  43. 43. Wagyu male Cow from Australia Wagyu female cow from Australia High quality bull High quality cow Cloning animal technique Cloning Animal Technique Tế bào từ lổ tai Animal with high quality of meat This research is funded by Vietnam National Project (2016-2021)
  44. 44. The process of using transgenic- cloned animals to produce human therapeutic proteins (Human drugs) 100.000.000 USD/Cow Animals Pharming
  45. 45. PHARMING for FARMACEUTICALS Classic method to produce human therapeutic proteins Novel method to produce human therapeutic proteins Human proteins producing genes Bacterial DNA Plasmid DNA Plasmid DNA cut with restriction enzymes Recombinant DNA Recombinant Bacterium Fermentation Tank Human proteins Human proteins Human proteins producing genes Positive, negative selection Negative (GFP-) Positive (GFP+) DNA Transfection Dairy cow cells Cloned transgenic embryo Embryos transfer Human proteins Cloning animal Transgenic cow
  46. 46. Supper dairy cow Great business in Animal Farming (Pharming) Therapeutic Proteins Animal $/animal/year AAT (alpha-1-antitrysin) Cow 150.000 USD/cow/year tPA (Tissue plasminogen activator, chống đông máu) Cow 700.000 USD/cow/year Factor VIII or IX or (Blood clotting factors, điều trị chống chảy máu). Cow 200.000 -300.000 USD/cow/year Lactoferrin 25.000 USD/cow/year CFTR (Cystic fibrosis transmembrane conductance regulator) Cow 800.000 USD/cow/year Human Protein C (Chống đông máu) Cow 10 milions USD/cow/year Fibrinogen (điều trị vết thương) Cow 400.000 USD/cow/year Glutamic acid decarboxylase (Điều trị tiểu đường type 1) Cow 200.000 USD/cow/year Anpha-lactalbumin (điều trị nhiễm trùng) Cow 500.000 USD/cow/year 100.000.000 USD/cow
  47. 47. PHARMING In 2009, we succeeded transgenic pig Human Erythropoietin (hEPO) gene, and that transgenic pig shown to have a normal reproductive ability 1 2 Konkuk University, Sep 24, 2009 Full-term development of human erythropoietin transgenic cloned pigs
  48. 48. Bio-organs for human? ? ? ? ? ? ? ? ? ? ? Bio-organs
  49. 49. Animals organs for human: Bio-organ Our group has succeed in making the korean's first 1,3-galactosyltransferase- knockout cloned miniatures pigs. This pig organs will be used for xenotransplantation into human without immune rejection . For Xenotransplantation (pig’s organs to human)
  50. 50. Mouse Pdx1 -/- (Blastocyst) Rat (ES cells) Chimeric mouse with rat pancreas Generation of RAT pancreas in Pdx1 knockout mice Kobayashi et al., Cell 2010 0ur hypothesis: Can we make human organ from animal bodys?? Animals organs for human: Bio-organ
  51. 51. Pig fibroblasts cells Pig enucleated oocyte siRNA Transfection Lentiviral vector Pig knockout pancreas, heart, liver, kidney related genes cells Organ related genes knockout cloned pig blastocyst Human iPS cells Embryo transfer Humans organs ? ? ? ? ? ? ? ? ?? Cloned pig with human organs Animals organs for human: Bio-organ
  52. 52. Business of 20 century Conclusion: SMART LIVESTOCK FARMING Business of 21 century Biotechnology for human SMART Livestock Farming for WATER security
  53. 53. Thank you for your attention Cellular Reprogramming Lab Members

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