PENGETAHUAN DASAR: KULTUR, MIKROMANIPULASI dan TRANSFER ... · PENGETAHUAN DASAR: KULTUR,...

PENGETAHUAN DASAR: KULTUR, MIKROMANIPULASI dan TRANSFER

INTI SEL (kloning)

Dr. Gatot Ciptadi

Pada suatu saat : Dreams comes true……

www.bankselgamet.com

Introduction

• DREAMS SOMETIMES COME TRUE !

Bioteghnology has the possibility to be revived a

scare and an extinct animal (Film Jurasic Park)

• The birth of the clone animals is great influencing

the frontier of research of animal reproductive

cells biotechnology

• Why ?

• Somatic cell (deferentiated) was

reprogramed back to embryonal stage

and resulted in normal offsprings.www.bankselgamet.com

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 transferDolly (February 1997)

Normal oocyte

Somatic

cell

Enucleated

oocyte

Cloning

277 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


Bagian II


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 r

Ukuran sel

S e l e k s i S e l Resipien sel

ASPEK TEKNIS


Treatments of activation Selected M-II Oocytes Cleavage Rate (%)

Control (M-II) 40 0.00

GCSE 2.5 ug/ml 74 6.00

7 % ethanol+GCSE 2.5 ug/ml 251 36.33

7 % ethanol + GCSE of 100 kDA 241 2.00

Table1. Cleavage rate of different treatments using CSE supplementation

Control GCSE GCSE2+ET

The result showed that the intensity of Ca+2 is diffefrent among

treatments


RESULT :Activation : Analysis of Ca++ Intensity (Fluo-3, CLSM)

Histogram Line series

The increase of Ca+2 intensity that equal to Ca+2 concentration,

might lead to egg activation. Oocytes activation was induced by

chemical agent supplemented with CSE, with the evidence of

rise Ca+2 intensity. This was considered to be sufficient factor for

activationwww.bankselgamet.com

DATA

FISIOLOGI

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, MIKROMANIPULASI dan TRANSFER ... · PENGETAHUAN DASAR: KULTUR,...

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