Somatic Cell Nuclear Transfer-new

Somatic Cell Nuclear Transfer

By: Prashant Kumar MIT-4th Yr

Contents 1. 2. 3. 4. 5. 6. 7. 8.

Introduction Sources of ES cells Therapeutic Cloning Nuclear Transfer Cell cycle Molecular Mechanisms Safety & Pitfalls Future Prospects

Introduction

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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.

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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.

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They are able to contribute widely in chimeras.

Sources of ES cells 2.

IVF Embryos

5.

Cloned Embryos

Therapeutic Cloning 

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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.

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Derive Histocompatible ES cell from the resulting blastocyst

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Derive disease specific ES cells for research and drug discovery

Nuclear Transfer  

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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.

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Skin explants were cultured for 3 weeks in DMEM plus 10% fetal calf serum at 37 C and 5% CO2.

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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 

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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.

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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.

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Gene with aberrant methylation pattern may not reveal in ES Cell line but may come in to play when differentiation induced.

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Poorly developed embryos.

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Loss of fetus through miscarriage.

Safety & Pitfalls   

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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 

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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 

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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