Somatic cell nuclear transfer is a technique used to produce cloned cells. In this procedure that is in somatic cell nuclear transfer, the nucleus from a somatic cell is transferred into an enucleated oocyte. This transfer helps in reprogramming of an enucleated oocyte into an embryonic cell.
Somatic cell nuclear transferred embryonic cells developmental potential is similar to that of normal embryonic stem cells. That is these cells can be maintained and used as pleuripotent stem cells.
Sources of Embryonic Stem Cells:
1. In vitro fertilized embryos can be used as a source of embryonic stem cells.
2. Cloned embryos can be used as a source of embryonic stem cells.
The goal of therapeutic cloning technique is to remove healthy adult cells from a patient, reprogramming the cells nuclei, then collecting these cells and growing in a culture media. Pleuripotent embryonic stem cell clones from blastocyte are cultures and then induced to differentiate these cells into stem cell or required type of mature cell to treat that particular disease.
Uses of Nuclear Transfer Technique:
1. Histocompatible embryonic stem cells are extracted from blastocysts.
2. Disease specific embryonic stem cells can be derived and used for research and also in drug discovery.
1. Women between the ages 24 and 32 years with at least one biological child are selected as egg donors.
2. Donors were also screened for infectious diseases such as hepatitis virus B, hepatitis virus C, human immunodeficiency virus and also for human T-cell leukemia virus.
3. Donor women ovaries were down-regulated by at least two weeks of oral contraceptives, and then it is followed by controlled ovarian hyper stimulation with injections of gonadotropin hormone.
4. When leading follicle reaches at least 18mm, then human chorionic gonadotropin was administered.
Somatic Cell Isolation:
5. Adult human fibroblast cells are isolated from skin biopsies.
6. These skin explants are then cultured for three weeks in a suitable media, such as Eagle's minimal essential medium along with 10% fetal calf serum at 37 degree temperature and 5% carbon dioxide.
7. Once cellular outgrowth was observed, fibroblasts and keratinocytes were dissociated using enzymes such as 0.25% trypsin and 1mM EDTA.
8. Eggs collected from the donor were incubated with bisbenzimide along with cytochalasin B in embryo culture media for about twenty minutes. All these alterations were made in human tubal fluid, buffered with HEPES.
9. Chromosomes were studied using inverted microscope with Hoffman optic and also epifluorescent ultraviolet light. These eggs were then enucleated using a piezo electric device.
10. Blunt needle with mercury near its tip is used to gently penetrate the region known as zona pellucida and chromosomes are removed from the eggs.
Egg Activation and Culture:
11. After 35-45 hours of exogenous stimulation of eggs with Human chorionic gonadotropin, eggs are then activated by incubating them with 5mM ionomycin for about four minutes and then with 2mM 6-dimethylaminopurine
12. On the fourth day of culture, eggs cleave and resemble embryos.
Reprogramming by Molecular Mechanism:
1. Nucleus is breakdown
2. Histones are removed
3. Correct epigenetic reprogramming
4. Remodelling of chromatin is done
5. Finally methylation of DNA is done.
1. This technique is not safe as perturbation of gene may cause dysfunctioning of cells
2. If the methylation of genes are done in aberrant pattern, it may not be revealed in embryonic stem cell line but may play when differentiation of these cells are induced.
3. Embryos will develop poorly
4. Chances of miscarriage of foetus is very high
5. Offspring may have some of the genetic syndromes.
6. Success rate is very less, that is may be 1%-4%
7. Cloned mice showed shorter life span
8. Cloned mice can also develop obesity in adult life.
9. Imprinted genes may express improperly
10. Deregulation of imprinted genes are also seen
11. Somatic cell nuclear transfer technique is unsuccessful in dogs, monkeys and also in rats.
12. Epigenetic changes may also lead to cancer.
1. This technique can also be used to produce cell types required for the replacement therapy.
2. This technique can also be used to produce cardiomyocytes, and these cells can also be used to replace damaged heart tissues.
3. Treatment for genetic diseases like Parkinson's disease, and Multiple Sclerosis.
4. Skin grafting.
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