Cloning is the creation of an exact genetic replica of a small segment of DNA, a cell or a whole organism.Cloning is a form of asexual reproduction which is widespread in nature. In single cell organisms and plants, it is an entirely normal process (division, vegetative reproduction),Animals can be cloned by embryo splitting or nuclear transfer. Embryo splitting involves bisecting the multicellular embryo at an early stage of development to generate "twins". This type of cloning occurs naturally and has also been performed in the laboratory with a number of animal species.
Artificial cloning - is created artificially in a laboratory Dolly, the cloned sheep produced in Scotland in 1997 (Nature, 385, 810- 13).
Types of cloning
Natural cloning - Identical twins and multiple births are an example of human clones that are created naturally.Artificial cloning - is created artificially in a laboratory Dolly, the cloned sheep produced in Scotland in 1997 (Nature, 385, 810- 13).
DNA cloning - is also known as recombinant DNA cloning , molecular cloning and gene cloning. DNA cloning refers to the process by which a fragment of DNA is transferred from one organism to a self-replicating genetic element such as a bacterial plasmid or a virus. Plasmids are self-replicating extra-chromosomal circular DNA molecules and can be used to make many copies of the gene. These genetic elements can then be inserted into a host cell of interest and the function of the gene of interest studied. Gene cloning is also important for the development of drugs and treatments such as in pharmacogenetics and gene therapy.
Reproductive cloning - is also called adult DNA cloning. The purpose is to produce a genetic duplicate of an existing or previously existing organism. Reproductive cloning refers to the process by which an animal is created which had the same nuclear DNA as a previously existing animal. This is accomplished by the removal of the DNA containing nucleus of an egg and its replacement by the DNA from another cell. The egg is then stimulated to divide and following a number of divisions it can be transferred into the uterus of a suitable female host until its birth.
Therapeutic cloning, or embryo cloning refers to the production of human embryos for research purposes. The goal of this is not to create cloned babies but to harvest the stem cells of the embryo that have the potential to develop into almost any cell in the body
First, the donor cells are grown under special conditions in culture. The number of cells can be increased by several orders of magnitude. It is also possible to make genetic modifications and to select just those cells in which the desired modification has occurred and multiply these up. These cells are then fused with an unfertilised egg from which the introduced nucleus can lead to the formation of an embryo. The embryos are then transplanted into sheep and lambs are born naturally. This technology could allow the production of genetically identical groups of animals which possess a desirable genetic trait.
In vitro fertilization - IVF is carried out in micro droplets of culture medium. Each micro droplet contain 10 oocytes and 1 million sperms per ml (one dose).
Insertion of Recombinant DNA into Embryo – recombinant DNA is inserted into embryos using the techniques of Calcium phosphate precipitation or microinjection or retroviral infection or particle gun delivery or electrophoration
In vitro oocyte maturation (IVM) – the embryos are grown in the culture medium from cleavage stage to morula stage.
Embryo Transfer Technology - Recipients are estrus synchronized with donor during the previous estrous cycle. Prostaglandin F2-alpha is commonly used to cause luteolysis. Surgical and non-surgical means are used in the transfers.
Gestation and Parturtion - In normal reproduction an animal produces about 4-5 offsprings in a lifetime. IVF technology can produce 50-80 offsprings in a lifetime.
Therapeutic cloning, or embryo cloning refers to the production of human embryos for research purposes. The goal of this is not to create cloned babies but to harvest the stem cells of the embryo that have the potential to develop into almost any cell in the body
Artificial cloning procedures
Embryo splitting – the blastomeres must be totipotent at 8-16 cell stage. The cell mass is cut with a glass knife dividing groups of blastomeres. One set of cells put into denuded zona pellucid. Identical twins result from embryo splitting. Embryo splitting changes neither the age nor the (toti-)potency of the cells used. The (two) embryos from the splitting are in the same stage of development, exactly the same age as the undivided embryo would have been and genetically completely identical.Technique of Nuclear transfer technology
The nucleus transfer technique is the transfer of the genetic program (the cell nucleus with the desired genetic material) from a totipotent blastomere to an unfertilised egg cell whose nucleus has previously been removed. This technique basically offers the possibility of replicating an adult individual and their genetic program.First, the donor cells are grown under special conditions in culture. The number of cells can be increased by several orders of magnitude. It is also possible to make genetic modifications and to select just those cells in which the desired modification has occurred and multiply these up. These cells are then fused with an unfertilised egg from which the introduced nucleus can lead to the formation of an embryo. The embryos are then transplanted into sheep and lambs are born naturally. This technology could allow the production of genetically identical groups of animals which possess a desirable genetic trait.
Cloning technology of sheep
The unfertilised eggs are flushed out of a sheep which has been induced to produce a larger than normal number of eggs. Previously a sample of tissue was from the udder of a six year old ewe was taken and cultured in a dish (Dolly 1). The cultured cells are starved to send them into a resting or quiescent state. A cell is placed beside the egg and an electric current used to fuse the couplet. The reconstructed embryo is put into culture and grows for seven days. Embryos which grow successfully are taken and transferred to a sheep which is at the the same stage of the oestrus cycle as the egg. The sheep becomes pregnant and produces a lamb after 21 weeks (Dolly).Transgenic Technology
An animal whose genetic composition has been altered to include selected genes from other animals or species by methods other than those used in traditional breeding.Methods of Producing Transgenics
Superovulation – The donor animals are treated with pregnant Mare Serum Gonadotropin (PMSG)/follicle stimulating hormone-like to increase number of developing follicles. They are also administered with human chorionic gonadotropin (hCG)/ luteinizing Hormone-like to induce ovulation.Ovulation occurs every 21 days ( Cow, horse) and 16 days (sheep, goat).In well managed domestic cattles, 8 – 10 eggs are superovulated. Antral ( graffian follicles) are collected by laproscopic surgery. Recovered follicles are allowed to grow in vitro. Slaughterhouse ovaries also often used in livestock.In vitro fertilization - IVF is carried out in micro droplets of culture medium. Each micro droplet contain 10 oocytes and 1 million sperms per ml (one dose).
Insertion of Recombinant DNA into Embryo – recombinant DNA is inserted into embryos using the techniques of Calcium phosphate precipitation or microinjection or retroviral infection or particle gun delivery or electrophoration
In vitro oocyte maturation (IVM) – the embryos are grown in the culture medium from cleavage stage to morula stage.
Embryo Transfer Technology - Recipients are estrus synchronized with donor during the previous estrous cycle. Prostaglandin F2-alpha is commonly used to cause luteolysis. Surgical and non-surgical means are used in the transfers.
Gestation and Parturtion - In normal reproduction an animal produces about 4-5 offsprings in a lifetime. IVF technology can produce 50-80 offsprings in a lifetime.
Applications
“Gene pharming", i.e. the use of transgenic animals to manufacture (human) proteins with therapeutic use, e.g. in their milk. The active ingredients from biogenetic manufacturing processes (such as insulin, blood factors or other human bodily substances) can be obtained in much purer form. Production of active ingredients can be on a large scale and relatively cheap. Hazards to people (pathogens) can be avoided as far as possible by careful testing of drugs.
Cloning could be used in producing transgenic animals as animal models for human diseases. Animal models are used to study the biochemical and physiological processes, human diseases and possible therapies. New drugs can be tested in animal models for their toxicity and pharmacological effect on humans.
Xenotransplantation is transplanting animal organs into humans. Cloning of "donor animals",are made with the desired genetic modifications using nucleus transfer. The alien animal organ will perform its function in the human recipient.
Animal cloning will increase the genetic knowledge of productive animals. Animal cloning technologies will make the "production" of transgenic animals with modified (agricultural) characteristics. The goals for gene transfer in livestock breeding in combination with cloning are: quality enhancement, gene pharming, boosting resistance to disease, and cost reduction.
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