pengetahuan dasar: kultur sel hewan, … · hewan, mikromanipulasi embrio dan transfer inti sel ......

PENGETAHUAN DASAR: KULTUR SEL HEWAN, MIKROMANIPULASI

EMBRIO dan TRANSFER INTI SEL

Dr. Gatot Ciptadi

www.bankselgamet.com

BIOTEKNOLOGI REPRODUKSI MAMPU MENYELAMATKAN

SUMBER GENETIK HEWAN LANGKA

GT

Implementasi Kultur Sel

(Hewan/Ternak)somatis

oosit

sperma


GT

PERKEMBANGAN BIOTEK-REPRO: IMPLEMENTASI DI BIDANG KEDOKTERAN

DAN SCIENCE


IVM: 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

Embrio/Sel

Stem cell Transgenic animal

Cloning/NT

Cryopresevation

Embryo sexing

Drug testing

Early

developmental

gene

Genome

reprogramming

Potential benefits ART (Human and Animal):


Klasifikasi:Cloning Techniques

Desection of morula and blastocyst (Embryo)

Deaggregation (Isolation) of blastomeres (Embryo)

Clone

Nuclear transfer Micromanipulation

Micromanipulation +Gene transfer

1. Desection of morula and blastocyst (Embryo)

2. Deaggregation (Isolation) of blastomeres (Embryo)


Somatic nuclear transfer

Dolly (February 1997)

Normal oocyte

Somatic

cell

Enucleated

oocyte

Cloning277 nuclear

transferred

29 implanted

1 live birthwww.bankselgamet.com

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)www.bankselgamet.com

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


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


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


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


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


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


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


EnucleatedNon enucleated

IDNI

TRANSFER NUKLUES ATAU

TRANSFER GEN www.bankselgamet.com

GTwww.bankselgamet.com

a) Embryo cloningProduce 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


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.


Newly formed embryo containing DNA from somatic cell

cell division

implant

(Diploid )


• 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


Culturing of pluripotent Stem Cell from fertilized embryo

(David Cameron, 2001)

Culturing of pluripotent Stem Cells from cloned embryo


Cel

l

repla

cem

ent

ther

apy

ES cells

Human embryonic stem cells

Thomson et al., 1998

Science. 282:1145-7


Have a nice dream………

Tanaka Hozumi.


pengetahuan dasar: kultur sel hewan, … · hewan, mikromanipulasi embrio dan transfer inti sel ......

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