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PENGETAHUAN DASAR BIOTEKNOLOGI: MIKROMANIPULASI EMBRIO,TEKNIK
TRANSFER INTI SEL
Dr. Gatot Ciptadi
Dr.G Ciptadi UB L7
Traditional vs. Modern Biotechnology
Traditional
• Based on microorganisms
• Cells are not manipulated
• Production of antibiotics by fermentation
Modern
• Based on microorganisms and cell culture
• Cells are manipulated
• Production of protein products based on recombinant DNA
and cell culture
• Modern biotech refers to Molecular Biology, Molecular
Genetics and Genetic EngineeringDr.G Ciptadi UB L7
Contoh: Animal Applications
•Animals can be used as bioreactors!
•Many human therapeutic proteins are needed in massive quantities (>100s of kgs), so scientists create female transgenic animals to express therapeutic proteins in milk.
• Goats, cattle, sheep, & chickens are sources of antibodies (protective proteins that recognize & destroy foreign material)
•Transgenic refers to containing genes from another sourceDr.G Ciptadi UB L7
GT
PERKEMBANGAN BIOTEK-
REPRO: IMPLEMENTASI DI BIDANG
KEDOKTERAN DAN SCIENCE
Dr.G Ciptadi UB L7
Cloning Techniques
Desection of morula and blastocyst (Embryo)
Deaggregation (Isolation) of blastomeres (Embryo)
Clone
Micromanipulation
Nuclear transfer
Micromanipulation +Gene transfer
1. Desection of morula and blastocyst (Embryo)
2. Deaggregation (Isolation) of blastomeres (Embryo)
Dr.G Ciptadi UB L7
Somatic nuclear transfer
Dolly (February 1997)
Normal oocyte
Somatic
cell
Enucleated
oocyte
Cloning277 nuclear
transferred
29 implanted
1 live birthDr.G Ciptadi UB L7
a) Embryo cloning
Produce mono zygotic twins or triplets
Fertilized embryo
remove
one/more cells
Encourage to develop into embryo
Twins /triplets have identical DNA
b) Reproductive cloning
Produce a duplicate of existing animal
Used to clone sheep & other mammals
Produce several genetic defects
Medical ethicists- Immoral procedure to be done on humans as it is
unsafe & unethical
Scientific technique to
create genetically
identical organisms
Cloning
1. Embryo Cloning
2. Reproductive cloning
3. Therapeutic Cloning
Dr.G Ciptadi UB L7
c) Therapeutic Cloning
Somatic Cell NT
Biomedical Cloning
Research Cloning
Regenerative Medicine
Nuclear Trans.Therapy(NTT)
DNA is extracted from a human’s cell
inserted into a woman’s ovum
develop and produce stem cells.
stem cells are removed from the pre-embryo
grown into specific organ
transplanted into the patient.
Dr.G Ciptadi UB L7
Newly formed embryo containing DNA from somatic cell
cell division
implant
(Diploid )
Dr.G Ciptadi UB L7
• Therapeutic cloning (research cloning) is when stem cells are
extracted to grow into a piece of human tissue which is encouraged
to grow into a human organ for transplant
Dr.G Ciptadi UB L7
Culturing of pluripotent Stem Cell from fertilized embryo
(David Cameron, 2001)
Culturing of pluripotent Stem Cells from cloned embryo
Dr.G Ciptadi UB L7
Cel
l
repla
cem
ent
ther
apy
ES cells
Human embryonic stem cells
Thomson et al., 1998
Science. 282:1145-7
Dr.G Ciptadi UB L7
Reduce both cost of of drug treatment and wastage of immature eggs
collecting during standart IVFCould also lessen the risk of hyperstimulation syndrome
May provide a valuable model for investigating
the causes of meiotic aberattions and aneuploidies
Preserved ovarian tissue
Might open to oocyte cryopreservation
Application of
IVEP/NT
Stem cell Transgenic animal
Cloning/NT
Cryopresevation
Embryo sexing
Drug testing
Early
developmental
gene
Genome
reprogramming
Potential benefits ART (Human and Animal):
Dr.G Ciptadi UB L7
Basic technique for nuclear transfer
(cloning)1. Cryopreservation (Freezing) Seeding, Stepwise, Direct, Vitrification
2. Embryo transfer (ET) Superovulation
Collection of embryos (Flushing)
Evaluation of embryos
Synchronization of estrus
3. In vitro fertilization (IVF) Collection of oocytes from ovaries
In vitro maturation
In vitro fertilization
In vitro culture
4. Micromanipulation Sperm injection (ICSI; IVM, Activation, IVC)
Clone (Nuclear transfer)
IVM, IVF, IVC
Enucleation, Nuclear injection
Fusion, Activation
Gene transfer ( vector, Electropolation)
Gene transfer (Injection into zygotes)Dr.G Ciptadi UB L7
In vitro fertilization (Animals)
Frozen semen
Thaw
In vitro sperm treatment and Capacitation
In vitro matured oocytes
Incubation with 1-2 million/ml
spermatozoa for 18 h
Presumptive zygotes washed with IVC media
Placed in 50-100 μl droplets of IVC media in 35 mm
Petri dish in groups of 10-15
Dr.G Ciptadi UB L7
IVF- Laboratory Processes (human)
Eggs retrieved
Eggs stripped and cleaned
Assess sperm
quality and count
Wash
sample
Sperm collection
Egg equilibration
Fertilization- IVF or
ICSI
Assess fertilization
Incubate
Wash/remove
excess sperm
Assess &
Transfer
Embryos
Dr.G Ciptadi UB L7
Nuclear transfer Activation
ET
Invitro maturation
Surrogate mother pig
S u m m a r y of NTRecipient oocytes
Donor cell
Culture of Cloned
embryos
In vitro cultureEnucleation
Dr.G Ciptadi UB L7
Introduction of donor cell
Enucleation
Passages of
somatic cells
Activation
Enbryo transfer
Production of transgenic piglets
In vitro Maturation
of oocytes
Introduction of
Extraneous gene
XenotransplatationProduction of
human protein
Synchronization
of estrus
Surrogate mother of miniature pigs
Nuclear trasfer
Recipient oocytesDonor cell derived
from miniature pigs
Selection of M2
Dr.G Ciptadi UB L7
In Vitro Maturation of Oocyte (Cell Recipient)
Material : Oocyte.-Cumulus-Granulosa Cell Complexes
from large antral follicles (4 – 6 mm)
Medium : TCM199, (10 % FCS, 0.1 ng/ml Na- pyruvate,
Antibiotics.
Hormon : FSH and LH
Temperatur : 38.5 – 39 0C
Gas % : 5 % CO2 in air
Dish : Falcon 35 mm
Drop : 100 ul ( 10 ul/oocyte), covered with
mineral oil/ parafin oil
Dr.G Ciptadi UB L7
Sato,E, 2006, Ciptadi,2005
In vitro Maturation of Oocytes:
1. Oocyte at the GV stage mature to M-II (oocyte maturation)
2. Maturation can be induced in vitro (IVM)
3. Oocyte maturation : cumulus expansion, emission 1 st PB
Dr.G Ciptadi UB L7
1
2
3
4
A B
Sel Hidup… / Mati…
1. Besar,
2. Sedang,
3. Kecil: 22.16 %
4.Abnormal
S e l e k s i S e l D o n o rUkuran sel
S e l e k s i S e l Resipien sel
Dr.G Ciptadi UB L7
Nuclear transfer procedure to produce
reconstructed embryo:
inject donor cell in the perivittelin space of enuclated oocyte
• the performed fusion cell is performed
• intracytoplasmic direct nuclear injection (IDNI)
• activated artificially
PERSIAPAN
MIKROMANIPULASI
1.ENUKLEASI
2.TRANSFER INTI
Dr.G Ciptadi UB L7
EnucleatedNon enucleated
IDNI
TRANSFER NUKLUES ATAU
TRANSFER GEN Dr.G Ciptadi UB L7
GTDr.G Ciptadi UB L7