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CLONING

CLONING

  • Cloning means making an identical copy of the clones (a plant or animal specimen) – physically as well as genetically. Clones are exact replica of plants or animals, replicated asexually.
  • A clone derives its genetic matter from a single parent. Cloning involves the removal of the nucleus from one cell and its placement in an unfertilized egg cell whose nucleus has either been deactivated or removed.
  • As every cell is equipped with a material (i.e. chromosome) & know how to build a full adult of the spaces. It is therefore possible to reproduce any organism if a living cell from any part of organism is available.
  • It was British biologist, J.B.S Haldane (1963) who for the first time theorized that it would be possible to produce genetic duplicates from all living organisms includes human beings.

MOLECULAR CLONING

  • Molecular cloning refers to the process of making multiple molecules.
  • It is commonly used to amplify DNA fragments containing whole genes, but it can also be used to amplify any DNA sequence such as promoters, non-coding sequences and randomly fragmented DNA.
  • It is used in a wide array of biological experiments and practical applications ranging from genetic fingerprinting to large scale protein production.
  • Cloning of any DNA fragment essentially involves four steps:
  1. fragmentation – breaking apart a strand of DNA
  2. ligation – gluing together pieces of DNA in a desired sequence
  3. transfection – inserting the newly formed pieces of DNA into cells
  4. screening/selection – selecting out the cells that were successfully transfected with the new DNA
  • Initially, the required DNA needs to be isolated to provide a DNA segment of suitable size. Subsequently, a ligation procedure is used where the amplified fragment is inserted into a vector (piece of DNA).
  • The vector (which is frequently circular) is linearised using restriction enzymes, and incubated with the fragment of interest under appropriate conditions with an enzyme called DNA ligase.
  • Following ligation the vector with the insert of interest is transfected into cells. A number of alternative techniques are available, such as chemical sensitization of cells, electroporation, optical injection and biolistics.
  • Finally, the transfected cells are cultured. As the aforementioned procedures are of particularly low efficiency, there is a need to identify the cells that have been successfully transfected with the vector construct containing the desired insertion sequence in the required orientation.
  • Modern cloning vectors include selectable antibiotic resistance markers, which allow only cells in which the vector has been transfected, to grow. Additionally, the cloning vectors may contain colour selection markers, which provide blue/white screening (alpha-factor complementation) on X-gal medium.
  • Nevertheless, these selection steps do not absolutely guarantee that the DNA insert is present in the cells obtained. Further investigation of the resulting colonies must be required to confirm that cloning was successful. This may be accomplished by means of PCR, restriction fragment analysis and/or DNA sequencing.

ANIMAL CLONING

  • Since 1990s, animal cloning has become a possibility. Dolly, a sheep was the first mammal to be produced through cloning method by a team of scientists led by Dr.lan Wilmut at Roslin Institute, Edinburgh, UK in February 1997, through nuclear transfer technology.

HUMAN CLONING

  • Human cloning is the creation of a genetically identical copy of an existing or previously existing human. The term is generally used to refer to artificial human cloning. There are two commonly discussed types of human cloning: therapeutic and reproductive cloning.
  • Reproductive Cloning is method of delivering a baby by transferring nucleus of an adult human cell to an enucleated human egg cell and allowing the manipulated egg cell to grow normally in the uterus of a surrogate mother.
  • Therapeutic Cloning is the method of using stem cells from the cloned human embryo to produce human organs and replacement tissues for medical purposes.
  • A third type of cloning called replacement cloning is a theoretical possibility, and would be a combination of therapeutic and reproductive cloning. Replacement cloning would entail the replacement of an extensively damaged, failed, or failing body through cloning followed by whole or partial brain transplant.
  • The first hybrid human clone was created in November 1998, by Advanced Cell Technologies. It was created from a man’s leg cell, and a cow’s egg whose DNA was removed. It was destroyed after 12 days.
  • Since a normal embryo implants at 14 days, Dr Robert Lanza, ACT’s director of tissue engineering, told the Daily Mail newspaper that the embryo could not be seen as a person before 14 days.
  • On January, 2008, Wood and Andrew French, Stemagen’s chief scientific officer in
  • California, announced that they successfully created the first 5 mature human embryos using DNA from adult skin cells, aiming to provide a source of viable embryonic stem cells.
  • Dr. Samuel Wood and a colleague donated skin cells, and DNA from those cells was transferred to human eggs. It is not clear if the embryos produced would have been capable of further development.

CLONING IN STEM CELL RESEARCH

  • Somatic cell nuclear transfer (SCNT) is used to create embryos for research or therapeutic purposes. The most likely purpose for this is to produce embryos for use in stem cell research. This process is also called “research cloning” or “therapeutic cloning.”
  • The goal is not to create cloned human beings (called “reproductive cloning”), but rather to harvest stem cells that can be used to study human development and to potentially treat disease. While a clonal human blastocyst has been created, stem cell lines are yet to be isolated from a clonal source.
  • Therapeutic cloning is achieved by creating embryonic stem cells in the hopes of treating diseases such as diabetes and Alzheimer’s. The process begins by taking out the nucleus that contains the DNA from an egg and putting it in a nucleus from an adult.
  • In the case of someone with Alzheimer’s disease, the nucleus from a skin cell of that patient is placed into an empty egg. The reprogrammed cell begins to develop into an embryo because the egg reacts with the transferred nucleus.
  • The embryo will become genetically identical to the patient. The embryo will then form a blastocyst which has the potential to form/become any cell in the body.

SCIENCE AND TECHNOLOGY

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