Biotechnology applications in agriculture can help improve water security. Advances in embryo technology, cloning, and transgenic animals can lead to smarter livestock farming practices. This includes using techniques like ICSI and embryo transfer to rescue rare and infertile animals or produce livestock with desirable traits. Somatic cell nuclear transfer can also be used to conserve species. Additional biotechnologies such as induced pluripotent stem cells may allow the production of human organs in animals through knockout of specific genes, helping regenerative biomedicine and pharmaceutical production. Overall, smart applications of biotechnology in agriculture can enhance sustainable development and water security.

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. 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. 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. 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. Why do we need the responsibility to protect WATER on
our earth
Drought Flood
We'll need to use water much more smartly

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. SMART Water use in Agriculture
In industry: SMART ENERGY In Agriculture: SMART Horticulture
SMART Livestock Farming??

8. In Agriculture: Livestock Farming System
SMART Livestock Farming??
SMART Water use in Agriculture

9. SMART Water Use in Livestock Farming
Traditional livestock farming
Smart livestock farming
Applications of high-
biotechnology to the farm

11. 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

12. Nguyen Van Thuan, Ph.D.
Department of Bioscience & Biotechnology
Konkuk University, Seoul, Korea
vanthuan@konkuk.ac.kr
Advance Embryo
Technology

13. Fertilization
Sperm
Egg
(oocyte)
Zygote 8-cell embryo
The first
differentiation
Blastocyst
Preimplantation development
What is the EMBRYO?

14. 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

15. Fertilization
Sperm
Egg
(oocyte)
Fertilization: beginning of new organism

16. Infertile Treatment: ICSI

17. Embryos transfer (ET)
Embryos with high quality
Supper dairy cow
ET

18. 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

19. Development of normal mice from oocytes
injected with freeze-dried spermatozoa
Instant sperm:
Just add water
Wakayama T, and Yanagimachi R. 1998.

20. Live/dead cell staining
Fresh sperm
After freeze-dried
Freeze-dried sperm were all died
Wakayama T, and Yanagimachi R. 1998.

21. 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

22. 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

23. Applications of ICSI
- Sperm preservation
- Embryo preservation
- Embryo transfer
- Transgenic Animals
- Treatment of infertile diseases
- etc…
Assisted Reproductive Techniques (ART)

24. 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

25. 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

26. 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.

27. 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

28. Somatic Cell Nuclear Transfer: Cloned Animals

29. 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

30. 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

31. 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

32. 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

33. Conservation of Infertile
and Rare Animals

34. 2.5 years old GFP-ICR male
Our unpublished data
Cloned GFP-ICR
Rescue infertile age male mouse via
Cloning Technique

35. Cloned female mice from donor cells of 3
year old ICR female by scriptaid treatment Our unpublished data

36. Next generations from male and female infertile
Rescue infertile age female mouse via
Cloning Technique
Our unpublished data

37. 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.

38. Application of Cloning Technique
Production of healthy cloned mice from bodies
frozen at -20 degrees C for 16 years.
Wakayama et al, PNAS 2008

39. 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

40. 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

41. Offspring derived from iPS cell
Reprogramming by iPS cell
iPS cells will inject into
tetraploid embryos. Can
we get offspring derived
from iPS cells???
??

42. 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

43. Somatic Cell Reprogramming:
Applications in high quality of
meat & Regenerative
Biomedicine

44. 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)

45. The process of using transgenic-
cloned animals to produce human
therapeutic proteins (Human drugs)
100.000.000 USD/Cow
Animals Pharming

46. 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

47. 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

48. 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

49. Bio-organs
for human?
?
?
?
?
?
?
? ? ?
?
Bio-organs

50. 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)

51. 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

52. 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

53. Business of
20 century
Conclusion: SMART LIVESTOCK FARMING
Business of
21 century
Biotechnology
for human
SMART Livestock Farming
for WATER security

54. Thank you for your attention
Cellular Reprogramming Lab Members