mikromanipulasi embrio,teknik transfer inti sel

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 Engineering
Dr.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)
Dr.G Ciptadi UB L7
•Transgenic refers to containing genes from another source
PERKEMBANGAN BIOTEKREPRO:
IMPLEMENTASI DI BIDANG
KEDOKTERAN DAN SCIENCE
Dr.G Ciptadi UB L7
GT
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
Normal oocyte
Enucleated
oocyte
277
29
1
nuclear
transferred
implanted
live birth
Somatic
cell
DollyCloning
(February 1997
Dr.G Ciptadi UB L7
a) Embryo cloning
Produce mono zygotic twins or triplets
Fertilized embryo
remove
one/more cells
Cloning
Scientific technique to
create genetically
identical organisms
1.
Embryo Cloning
Encourage to develop into embryo
2.
Twins /triplets have identical DNA3.
Reproductive cloning
Therapeutic Cloning
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
unsafe & unethical
Dr.G Ciptadi UB L7
on humans as it is
c) Therapeutic Cloning
Somatic Cell NT
DNA is extracted from a human’s cell
Biomedical Cloning
Research Cloning
inserted into a woman’s ovum
Regenerative Medicine
Nuclear Trans.Therapy(NTT) 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
(Diploid )
Newly formed embryo containing DNA from somatic cell
cell division
Dr.G
Ciptadi UB L7
implant
• Therapeutic cloning (research cloning) is when stem cells are
extracted to grow into a piece
of human
Dr.G Ciptadi
UB L7 tissue which is encouraged
to grow into a human organ for transplant
Culturing of pluripotent Stem Cell from fertilized embryo
(David Cameron, 2001)
Culturing of pluripotent Stem Cells from cloned embryo
Dr.G Ciptadi UB L7
Cell
replacement
therapy
Human embryonic stem cells
ES cells
Thomson et al., 1998
Science. 282:1145-7
Dr.G Ciptadi UB L7
Potential benefits ART (Human and Animal):
Stem cell
Genome
reprogramming
Early
developmental
gene
Drug testing
Transgenic animal
Cloning/NT
Application of
IVEP/NT
Embryo sexing
Cryopresevation
Reduce both cost of of drug treatment and wastage of immature eggs
collecting during standart IVF
Could also lessen the risk of hyperstimulation syndrome
May provide a valuable model for investigating
the causes of meiotic aberattions and aneuploidies
Preserved
ovarian tissue
Dr.G Ciptadi UB L7
Might open to oocyte cryopreservation
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)
Dr.GGene
Ciptaditransfer
UB L7 (Injection into zygotes)
In vitro fertilization (Animals)
Frozen semen
In vitro matured oocytes
Thaw
In vitro sperm treatment and Capacitation
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
Sperm collection
Eggs stripped and cleaned
Wash
sample
Egg equilibration
Fertilization- IVF or
ICSI
Assess sperm
quality and count
Wash/remove
excess sperm
Assess fertilization
Dr.G Ciptadi UB L7
Incubate
Assess &
Transfer
Embryos
S u m m a r y of NT
Recipient oocytes
Invitro maturation
Enucleation
Donor cell
Nuclear transfer
In vitro culture
Activation
Culture of Cloned
embryos
Surrogate mother pig
ET
Dr.G Ciptadi UB L7
Nuclear trasfer
Recipient oocytes
Donor cell derived
from miniature pigs
Activation
In vitro Maturation
of oocytes
Passages of
somatic cells
Surrogate mother of miniature pigs
Synchronization
of estrus
Selection of M2
Enbryo transfer
Introduction of
Extraneous gene
Production of transgenic piglets
Enucleation
Xenotransplatation
Dr.G Ciptadi
UB L7
Introduction of donor cell
Production of
human protein
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
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
Sato,E, 2006, Ciptadi,2005
Seleksi Sel Donor
Ukuran sel
1
2
3
4
B
A
1. Besar,
2. Sedang,
Sel Hidup… / Mati…
3. Kecil: 22.16 %
4.Abnormal
S e l e k s i S e l Resipien sel
Dr.G Ciptadi UB L7
PERSIAPAN
MIKROMANIPULASI
1.ENUKLEASI
Nuclear transfer procedure to produce
reconstructed embryo:
2.TRANSFER INTI
inject donor cell in the perivittelin space of enuclated oocyte
• the performed fusion cell is performed
• intracytoplasmic direct nuclearDr.G
injection
(IDNI)
Ciptadi UB
L7
• activated artificially
Non enucleated
Enucleated
IDNI
TRANSFER NUKLUES ATAU
TRANSFER GEN Dr.G Ciptadi UB L7
Dr.G Ciptadi UB L7
GT