Somatic Cell Nuclear Transfer-New

Somatic
Cell Nuclear Transfer
By:
Prashant Kumar
MIT-4th Yr
Contents
1. Introduction
2. Sources of ES cells
3. Therapeutic Cloning
4. Nuclear Transfer
5. Cell cycle
6. Molecular Mechanisms
7. Safety & Pitfalls
8. Future Prospects
Introduction
 In somatic cell nuclear transfer (SCNT), the nucleus of a

somatic cell is transferred to an enucleated oocyte for
reprogramming to an embryonic cell state.
 NTES cells are identical in their development potential to

normal ES cells in that they can be maintained as
pleuripotent stem cells when propagated in presence of LIF.
 They are able to contribute widely in chimeras.

Sources of ES cells
2. IVF Embryos
5. Cloned Embryos
Therapeutic Cloning
 Therapeutic cloning is a stem cell therapy strategy that
aims to combine CNR, human stem cell culture and
stem cell therapy.
 Its goal is to remove healthy adult cells from a
patient, reprogramme the cell’s nuclei by CNR, collect
and grow pluripotent embryonic stem cell clones
from the resulting blastocyst and then induce these to
differentiate into the stem cell or mature cell types
required for transplantation to treat disease.
Therapeutic Cloning
 Extension of Nuclear Transfer
Technique.
 Derive Histocompatible ES cell from

the resulting blastocyst
 Derive disease specific ES cells for

research and drug discovery
Nuclear Transfer
 Egg donors were women between the ages of 24 and

32 years with at least one biologic child.
 They were also screened carefully for infectious
diseases, including hepatitis viruses B and C, human
immunodeficiency virus, and human T-cell leukemia
virus.
 Donor ovaries were down-regulated by at least 2 weeks
of oral contraceptives, followed by controlled ovarian
hyper stimulation with twice daily injections of 75–150
units of gonadotropins.
 hCG was administered when the leading follicle
reached at least 18 mm by ultrasound examination.
Nuclear Transfer
Somatic cell isolation

 Adult human fibroblasts were isolated from 3-mm skin
biopsies.
 Skin explants were cultured for 3 weeks in DMEM plus

10% fetal calf serum at 37 C and 5% CO2.
 Once cellular outgrowth was observed, fibroblasts and

keratinocytes were enzymatically dissociated using
0.25% trypsin and 1 mM EDTA.
Nuclear Transfer
Egg enucleation and NT
 Eggs were incubated with 1 mg/ml bisbenzimide and
cytochalasinB in embryo culture media for 20 min. All
manipulations were made in HEPES buffered human tubal
fluid.
 Chromosomes were visualized with an inverted microscope
equipped with Hoffman optic and epifluorescent ultraviolet
light. Enucleation was performed using a piezo electric
device
 A 10 mm I.D. blunt needle that contained mercury near its tip
to be able to control the penetration capacity and accuracy of
the procedure was used to penetrate gently the zona pellucida
and aspirate the chromosomes.
Nuclear Transfer
Egg activation and culture
 At 35–45 h after exogenous hCG stimulation, eggs were

activated by incubating them with 5 mM ionomycin for 4 min
followed by 2 mM 6-dimethylaminopurine.
 On the fourthday of culture, cleaving eggs resembling

embryos .
DOLLY
Molecular Mechanisms
For Reprogramming:-
 Breakdown of nucleus.
 Removing the Histones.
 Correct Epigenetic reprogramming
 Chromatin Remodeling
 DNA methylation
Safety & Pitfalls
 Not safe:-Perturbations of a gene that could cause cell
dysfunction.
 Gene with aberrant methylation pattern may not reveal

in ES Cell line but may come in to play when
differentiation induced.
 Poorly developed embryos.
 Loss of fetus through miscarriage.

Safety & Pitfalls
 Large offspring syndrome.

 Success rate 1% -4%.
 Cloned mice have shorter life span and can develop
obesity in adult life.
 Improper expression of imprinted genes.
 Dysregulation of non imprinted genes.
 SCNT unsuccessful in dogs,monkeys and rats.
 Epigenetic changes may lead to cancer.
Future prospects
 Tran differentiation into cell types for replacement

therapy.
 Clinical application may include the production of
cardiomyocytes to replace damaged heart tissue or
insulin producing B-cells for patients with diabetes.
 Treatment for Parkinsons disease,Multiple Sclerosis.
 Replacement Therapy.
 Skin Grafting
Future prospects
 There is also the possibility that these cells could be
used to reconstitute more complex tissues and organs,
including blood vessels, myocardial “patches,”
kidneys.
 NT techniques have the potential to eliminate the
immune responses associated with the transplantation
of these various tissues, and thus the requirement for
immunosuppressive drugs and/or immunomodulatory
protocols that carry the risk of serious and potentially
life-threatening complications

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Somatic

Cell Nuclear Transfer

 In somatic cell nuclear transfer (SCNT), the nucleus of a

 NTES cells are identical in their development potential to

 They are able to contribute widely in chimeras.

 Derive Histocompatible ES cell from

 Derive disease specific ES cells for

 Egg donors were women between the ages of 24 and

Somatic cell isolation

 Skin explants were cultured for 3 weeks in DMEM plus

 Once cellular outgrowth was observed, fibroblasts and

Egg activation and culture

 At 35–45 h after exogenous hCG stimulation, eggs were

 On the fourthday of culture, cleaving eggs resembling

 Removing the Histones.

 Correct Epigenetic reprogramming

 Gene with aberrant methylation pattern may not reveal

 Poorly developed embryos.

 Loss of fetus through miscarriage.

 Large offspring syndrome.

 Tran differentiation into cell types for replacement

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