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