1a BXN TNg2013 Slidedaidien
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Bui Xuan Nguyen,Ph.D- University Paris VIVAST, USTH Hanoi, VietnamBuixn(@gmail.com
Cng ngh Phi & T bo gcEmbryo & Stem cells
Biotechnology- TM LC
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Embryo
-Etymology : First attested in English in the mid-14c., theword embryon derives from Medieval Latin embryo,itself from Greek (embruon), plural (embrua), lit. "young one
- An embryo is a multicellular diploid eukaryote in itsearliest stage of development, from the time of first celldivision until birth, hatching, or germination. In humans,it is called an embryo until about eight weeks after
fertilization and from then it is instead called a fetus.(wikipedia]
- Parthenogenetic haploide, trysomy ???
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Embryo development
- Embryogenesis: The development of the embryo
- Embryo differentiation: The process by whichspecialized and diversified structures arise duringdevelopment of the embryo. The process involves (1) anincrease in the number of cell types, and (2) anincrease in morphological heterogeneity through the
arrangement of cells into increasingly complexstructural patterns in the form of tissues and organs.(wikipedia]
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EMBRYO
Fetus
New organism
OocyteSperm
Embryo- the start in life- all begins from embryo
4 groups connective,muscle, nervous &epithelial tissues
300 different cell types
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Embryo differentiation
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Stem cell key properties
Principles of stem cell biology
Self-renew & multiply Differentiation , Plasticity
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General approaches for stem cell production
1. Adult Stem Cells
2. Embryonic Stem Cells (ES)
3. Induced Pluripotent Stem Cells (iPS)
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EMBRYO
New organism
OocyteSperm
Fertilization, Embryo, Stem cells & Development
Cells
Totipotent
Pluripotent
Multipotent
Unipotent
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2. Embryonic stem cells ( pluripotent)
From in vitro fertilizedembryos (IVF):
Mouse (Evan & Kaufman,1981); Human (Thomsonet al, USA, 1998/,
Sweden, UK, Japan,China, Korea, Spain, Iran,Israel, Singapore,Thailand)
From Somatic Cell Nuclear Transfer (SCNT) embryo: Mouse(Wakayama et al, 2001), Bovine, Monkey, Pig, ( Human: Sweden,
China)
From Parthenotes
( parthenogenetically activated oocytes ): mouse (Cuthbertson,1983),human (Cibelli et al. 2001).
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1.Adult stem cells (multipotent)
Umbilical cord matrix stc :
Cord Is Richer than Blood:0.7-2.0 x106cells/cm( Secco et al, 2007)low immunogenicity, lower expression of CD106, HLA-ABC and HLA-DR, benefit clinical use. (Lu et al, 2006)
"stem cell niche" in brain,bone marrow, peripheralblood, blood vessels,skeletal muscle, skin,teeth, heart, gut, liver,ovarian
Photo: STEMCELL Technologies Inc
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3- Induced Pluripotent Stem Cells
First: Takahashi v Yamanaka (2006): exogenous expressionof the transcription factors OCT4, SOX2, KLF4, & c-MYCNew method single transfer of ESC (embryonic stem cell)-derived proteins into fibroblasts, full reprogramming up to thepluripotent state (Chio et al, 2010).
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TECHNOLOGY
BIODIVERSITY PROTECTION
TRANSGENICPHARMACEUTICALINDUSTRY
REGENERATION MEDICINE
XENOTRANSPLANTATION
GENE THERAPY
INFERILITY TREATMENT
PGD
ANIMAL BREEDING INDUSTRY
BIO-MEDICAL APPLICATIONREPRODUCTION
Superovulation
In vitro Fertilization
Somatic cloning
XX
IVM-IVF38 0C, 5% CO2
Genetic Engineering
Stem cells
Cryobanking
Embryo Transfer
BXNguyen-Paris VI
EMBRYO
New organism
OocyteSperm
Cells
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The fusion of biology and technology. Biotechnology isthe application of biological techniques to productresearch and development. (http://www.medterms.com/)
Biotechnology: Definition
A field of applied biology that involves the use of livingorganisms and bioprocesses in engineering, technology,medicine and other fields requiring bioproducts.(http://en.wikipedia.org/wiki/Biotechnology)
Any technological application that uses biologicalsystems, living organisms, or derivatives thereof, tomake or modify products or processes for specific use.(TheUnited Nations Convention on Biological Diversity)
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Classic Biotechnology arose from the field ofzymotechnology, which began as a search for a betterunderstanding of industrial fermentation
Biotechnology: Definition
A
Modern Biotechnology: The origins of biotechnologyculminated with the birth of genetic engineering.-the 1953 discovery of the structure of DNA, by Watsonand Crick- 1973 discovery by Cohen and Boyer of a recombinant
DNA technique by which a section of DNA was cut fromthe plasmid of an E. coli bacterium and transferred intothe DNA of another
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Technology
Bio-Medical BiotechnologyAt central first life stage for alllife aspects
Embryo &Stem cell
Life application
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SCNT- 1. Production of genetic identical stem cells
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EMBRYO & STEM CELLS TECHNOLOGY
W Heape, 1890: first animal fromembryo transfer
Edward, 1978: first test-tube baby
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a dng sinh hc,cong ngh sinhsn- bo tn ex
situ-in situ
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Trong khong 440 triu nm qua c 5 ln sinh vtb tiu dit hng lot
440 T, nguyn nhn ?
370 T, nguyn nhn ?
240 T, nguyn nhn ni la
202 T, nguyn nhn ?
65 T, nguyn nhn Sao chi
(V Qu, Biodiva 2007)
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Ln gn y nht, cch nay khoang65 triu nam l do thin thch rivo tri t, 20 % s h v 50% sging b tiu dit, trong c cc
loi khng long
Hin nay, ang din hin t-ng cc
loi b tiu dit hng lot, nh-ngnguyn nhn chnh l do hot ngca loi ng-i.
(V Qu, Biodiva 2007)
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Kt qu nghin cu ca hn 1500 nh
khoa hc trn th gii trong bnMillennium Ecoystem Assessment(2005) th trong khong 400 nm qua c 484 loi ng vt v 654 loi thc
b tiu dit, v c khong 30000 loi Vv TV ang nguy cp.
Tc cc loi b tiu dit nhanh gp
1001000 ln so vi bnh th-ng.Trong cc thp k sp ti tc mtcc loi cn cao hn.
(V Qu, Biodiva 2007)
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Danh lc Sch ca IUCN (2004):
23% s loi th (1101 loi) 12% s loi chim (1213 loi)
ang nguy cp cc nhm khc khong 38% n86% s loi nguy cp.
T 1970 n nay c 58 loi c, 9loi chim v 1 loi th b tiu dit.
(V Qu, Biodiva 2007)
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Nguyn nhn suy thoi cc loi:
1 Ni sinh sng b thu hp li2 Xm ln ca cc loi ngoi lai3 nhim mi tr-ng
4 Sn bt qu mc5 Thay i kh hu ton cu.
(V Qu, Biodiva 2007)
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Brief history of embryo freezing
1949 : Polge et al., the successful freezing of chickensperm by including glycerol
1952: Marden sent rabbit embryos by air back toCambridge from Worcester, packed with iceballoons in a vacuum flask.
1960: Successful freeze-drying of bull sperm by Meryman1972: mouse embryos were successfully frozen &
transferred to give live young (Whittingham et al.,).
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Status of oocyte-embryo cryoconservation:
Technical aspects
Slow freezing (Wittinghqm et al, 1972).
Rapid freezing (Nguyen-JP Renard 1983)
Vitrification (Rall & Fahy, 1985).
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RT
-196oC
-30oC
Clacsic slowprogramable freezing
5 30 300-500
Rabbit, cattleSurvival in vitro: 60-88%In vivo: 33-81%
Sucrose-PROH, 2 min,Room temperature
Rapid freezing
(Nguyen, Renard,1983, 1984)
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Cloning bySomatic cell nuclear transfer /SCNTCloning byCloning by
Somatic cell nuclear transfer /Somatic cell nuclear transfer /SCNTSCNT
1997, Doily
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clone a woolly mammoth in the next five years.
Led by Akira Iritani, Kinki
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SCNT : conservation
Wells et al, 1999: Enderby island cattle
Robert et al, 2000, Gaur
Smith et al, 2001.
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American BisonAmerican Bison -- BisonBisonbisonbison
European BisonEuropean Bison -- BisonBisonbonasusbonasus AurochAuroch -- BosBosprimigenusprimigenus Wild YakWild Yak -- BosBosmutusmutus BantengBanteng -- BosBosjavanicusjavanicus
GaurGaur -- BosBosgaurusgaurus KoupreyKouprey-- BosBos SauveliSauveli African BuffaloAfrican Buffalo -- SyncerusSynceruscaffercaffer Wild Water BuffaloWild Water Buffalo -- BubalusBubalusarneearnee
LowlandLowland AnoaAnoa -- BubalusBubalusdepressicornisdepressicornis MountainMountain AnoaAnoa -- BubalusBubalusquarlesiquarlesi TamarawTamaraw -- BubalusBubalusmindorensismindorensis SaolaSaola -- PseudoryxPseudoryxnghetinhensisnghetinhensis
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Kouprey : Numbers estimated- before XX 2000- 1938 800- 1940 1000- 1970 30-70- 1975 50
- 1986 >200- 2003 >250 (IUCN 2004)
(V Qu, Biodiva 2007)
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Saola protection zone1. Vu Quang, 1995
2. PuMat- Lao-VN, 19953. BachMa, 20084. Quang tri, 2010
Nombre et superficie des aires protges au Vietnam
0
500,000
1,000,000
1,500,000
2,000,000
2,500,000
3,000,000
Year 1954 Year 1975 Year 1986 Year 1993 Year 2003
Ha
0
20
40
60
80
100
120
140
Sites
Superfice totale (ha) Nombre des aires protges
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1994 - Endangered (Groombridge 1994)1996 - Endangered (Baillie and Groombridge
1996)2003 - Endangered (IUCN 2003)2006 - Critically Endangered (IUCN 2006)
The saola(Pseudoryx nghetinhensis)
New discovered,
but already threathened
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NHAN BAN VO TINH- IN VITRO DEVELOPMENT OF BOVINE-BOVINEAND SAOLA-BOVINE EMBRYOS
Bovine-bovine SCNT D.8 Saola-Bovine SCNT D.8
Nbre cell. totales Nbre cell. ICM Nbre cell. tropho.
88 36 (40.9) 52 (59,1)
Donor cell
Cattle
Blastocyst (%)
Saola
21 (20.07)
46 (27.71)
Oocyte Cleaved (%) Morula (%)Fusion
150
312
91 (60.67)
166 (50.21)
72 (79.12)
131 (78.92)
25 (27.5)
65 (39.15)
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animals
Equip Mobil
Informaticconnection
REGIONAL & COUNTRY EX SITU-INSITU NETWORK
In situ: reservation1,2..
reintroduction
Country Central
Rescue station
Samples: semen, egs, cells
Cryobanking
Software
ART
Bio-Medicalapplication
InternationalRegional
Banking
Buffer ZoneEconomic Support
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CNSS-Ci to nhnging b tht-sa
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VAST Vietnam-Cc mc thnh cng u tin
1978
2000
1988
1986
1983
Chng trnh m ha
Lm ch rng trng trn tru
B cy phi
Phng php ng lnh nhanh phi
Th cy phi u tien
Nhn bn v tnh: b, kh
Xc nh gii tnh phi
Th tinh ng nghim
T bo gc phi nhn bn
2001
19991996
1994
2003
D cy phi
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Vn : kh nng phc v cy phi quy m 30-60 phi/ngy, a
im xa 70-2000 km)
Ch tiu: Thi gian Vi phu thut phi, t l phi sng , thi gian PCR,t l xc nh, mc chnh xc2 t hp mi : INRA1-148bp-c, INRA2-443 bp-loi,
BRY4a-300bp-c, STS -216bp-loi
ci ct bo phi
PCR
Primer ciPrimer c
sRY
(2)
(3)Vi phu thut
(1)
NST Y
Chn phi
Cy phi
Ni dung 2: Sn xut phi cao sn c gii tnh xc nh bngk thut PCR
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phi
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phi PROGESTERON
PG
PGFSH
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Estrus-superovulation synchronisation-ET
PG2PG1 DDuc1 hCG PG3PMSG/ FSH EmbryoAIDduc2
PG2PG1 DDuc1 hCG PG3PMSG/ FSH ETDduc2
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So snh t l cha do cy phi VPT-gii tnh vphi khng VPT
Loi phi S bnhn
(n)
Cha 3thng
n (%)
n (%)
B con B ci
n ( %)
GT 16 11 (68,8) 8 (50,0) 11 9 (81,8)
Khng GT 20 13 (65,0) 9 (45,0) 15 6 (40,0)
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Tng th
T l cha chun : > 50 %T l cha do cy phi GT: > 50%S b sinh ra : 41S b ci: 31
T l b ci do cy phi GT: 80%T l b sinh i do cy 2 phi: 50%Thi gian mang thai: 258- 275 ngyTrng lng s sinh: 22-50 kgTc pht trin: tng ng Holstein
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B TTON u tin Vietnam, Vinh Tuong, 11.2002
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B sinh do cy phi TTON v xc nh gii tnh u tin Vit nam, 2-2003
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so snh s b sa x tiu th sa/ dn s
VN: 32 cc/per
Thi Lan: 250
Php:5000
50000 200000 1.5 tr
2000 2010 ???
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300 000 animals
300
30
1 SCNT
SCNT for animal breeding through ONBS
Sexing
A.I
Embryo
30 years
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Xc nh giitnh:VPT phI+PCR
B csa+Nhit i
Phi cI
B H-n Top: B Holstein >8000 Lit/ck
Tip cn 2- cng ngh phi TTON
TINHTRNG
XX
T T O N38 0C, 5% CO2
cy phi vo b LaiSind
(1)
(2)
(3)
(4)
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H sinh sn nhn ging M
Ngun Gen Trng Tinhcao sn M, Nht
Cng nghip PhiTh tinh ng nghim
Ngun Gen Tinhhng nhit i
Dc, cichn lc Sib
c gingphn phi
Qungcanh
Qungcanh
Qungcanh
Qungcanh
Qungcanh
Qungcanh
Cy phi - nthng phm
Cy phi - nthng phm
n gck tip
Cy phi-n gc 1
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GENETIC & EPIGENETICASPECTS
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Inheritance & differentiation
Hypothesis of preformationism,N Hartsoeker, 1694: all organsWere prefigured whithin thesperm
Hypothesis of epigenesis,Each organism develops anew from anundifferentiated condition.
- each differentiated cell retain the fullcomplement of genetic informationpresent initially in the zygote? HansSpeman, 1938
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Hans Spemann (1869-1941)
1938 the "fantastical experiment" ofcloning by nuclear transfer
Nobel award 1935 for discovery ofthe organizer effect in embryonicdevelopment";
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Stem cell, Life creation-
one or two directions
Direction : gametes-embryo-cells
Direction : cells-embryo-gametes
Cytogenetic aspects?Molecular aspects?
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Shinya Yamanaka et al
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Ca
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Genomic RNA profiling and the programme controlling preimplantationmammalian development , Christine E. Bell et all, 2008
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Epigenetic regulation in mammalian preimplantationembryo development, Lingjun Shi and Ji Wu, 2009
General view of the main epigenetic reprogramming pathways in preimplantationdevelopment. (A) DNA methylationreprogramming in the mouse embryo. Active demethylationhappens in paternal PN(pronucleus) followed by passive demethylation of the entire genomeduring cleavage stage.
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X- inativation
Mechanisms from initiation of XCI to the shortandlong-term maintenance of silencing status. After selection, Xist coats the Xichromosome to initiate XCI. A ncRNA (RepA)then usher polycomb repressive complex 2 (PRC2) to the Xi chromosome, recruiting
histone modification changes such asH2A-K119 ubiquitination (U) and H3K27 methylation (M). Histone modifications bringabout short-term silencing followed byhistone variant incorporation (macroH2A) and de novo DNA methylation, mediatinglong-term silencing
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Direction : gametes-embryo-cells
Direction : cells-embryo-gametes
Oocyte & embryo -IVM-IVF IVF stc
SCNT embryo & embryonic SCNT stc Parthenogenetic Stc iPSc Cancer cells
Gen expresion, genetic &
epigenetic regulation