This article provides you with the basic biological knowledge of the process of cloning. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms.
Your knowledge of this technique will expose you to the limitless possibilities of cloning of animal/plant species.
In biology, cloning is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually.
Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms.
The term also refers to the production of multiple copies of a product such as digital media or software.
The term clone is derived from the Ancient Greek word (klon,”twig”), referring to the process whereby a new plant can be created from a twig. In horticulture, the spelling, clon was used until the twentieth century; the final “e‟ came into use to into use to indicate the vowel is a “long O” instead of a “short O”.
Since the term entered the popular lexicon in a more general context, the spelling, clone, has been used exclusively.
Molecular Cloning
Molecular cloning refers to the process of making multiple molecules. Cloning 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 finger printing to large scale protein production.
Steps in Cloning
Cloning of any DNA fragment essentially involves four steps:
Fragmentation – breaking apart a strand of DNA
Ligation – gluing together pieces of DNA in a desired sequence
Transfection – inserting the newly formed pieces of DNA into cells.
Screening/Selection – selecting out the cells that were successfully transferred with the new DNA.
Although these steps are invariable among cloning procedures, a number of alternative routes can be selected, these are summarized as a “cloning strategy‟.
Initially, the DNA of interest 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 under appropriate conditions with an enzyme call 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. 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. But this may not necessarily guarantee that the DNA insert is present.
Cell Cloning
Cloning a cell means to derive a population of cells from a single cell. In the case of unicellular organisms such as bacteria and yeast, this process is remarkably simple and essentially only requires the inoculation of the appropriate medium.
However, in the case of cell cultures from multi-cellular organisms, cell cloning is an arduous task as these cells will not readily grow in standard media.
Cloning Stem Cells
Somatic cells nuclear transfer, known as SCNT, can also be 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 diseases.
While a clonal human bastocyst has been created, stem cell lines are yet to be isolated from a clonal source.
Organism Cloning
Organism cloning (also called reproductive cloning) refers to the procedure of creating a new multi-cellular organism, genetically identified to another. In essence, this form of cloning is an asexual method of reproduction, where fertilization or inter-gamete contact does not take place.
Asexual reproduction is a naturally occurring phenomenon in many species, including most plants and some insects. Scientists have made some major achievements with cloning, including the asexual reproduction of sheep and cows.
There is a lot of ethical debate over whether or not cloning should be used. However, cloning or asexual propagation has been common practice in the horticultural world for hundreds of years.
Horticultural
Many trees, shrubs, vines, ferus and other herbaceous perennials form clonal colonies naturally. Parts of an individual plant may become detached by fragmentation and grow on to become separate clonal individuals.
Parthenogenesis
Clonal derivation exists in nature in some animal species and is referred to as parthenogenesis (reproduction of an organism by itself without a male). This is an asexual form of reproduction that is only found in some insects, crustaceans and lizards.
The growth and development occurs without fertilization by a male. In plants, parthenogenesis means the development of an embryo from an unfertilized egg cell, and is a component process of apomixis.
In species that use the XY sex-determination system, the offspring will always be female. An example is the “Little Fire Ant” (Wasmanniaauropunctata), which is native to Central and South America, but has spread throughout many tropical environments.
Species Cloned
Dolly the Sheep – Dolly, a Finn-Dorset ewe was the first mammal to have been successfully cloned from an adult cell. Dolly was formed by taking a cell from the udder of her biological mother. Her embryo was created by taking the cell and inserting it into a sheep ovum.
The embryo was then placed inside a female sheep that went through a normal pregnancy. She was cloned at the Roslin Institute in Scotland and lived there from her birth in 1996 until her death in 2003 when she was six.
The modern cloning techniques involving nuclear transfer have been successfully performed on several species. Notable experiments include:
Tadpole (1952)
Robert Briggs and Thomas J. King had successfully cloned northern leopard frogs: thirty-five complete embryos and twenty-seven tadpoles from one hundred and four successful nuclear transfers.
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Mice (1986)
A mouse was successfully cloned from an early embryonic cell. Soviet Scientists Chaylakhyan, veprencev, Svinidova, and Nikitin had the mouse “masha” cloned. Research was published in the magazine “Biofizika” volume XXXII, issue 5 of 1987.
Cattle: Alpha and Beta (males, 2001) and (2005) Brazil.
Camel (2009): Injaz, is first cloned camel.
Pashmina goat (2012)
Noori, is the first cloned pashmina goat. Scientists at the faculty of veterinary sciences and animal husbandry of sher-e-kashmir University of Agricultural Sciences and Technology of Kashmir successfully cloned the first pashmina goat (Noori) using the advanced reproductive techniques under the leadership of Riaz Ahmad Shah.
Others
Other species cloned include; Carp (1963), Sheep (1984), Rhesus Monkey (2000), Gaur (2001), Cat (2001), Rat (2003), Mule (2003), Horse (2003), Dog (2005), Wolf (2005),
Water buffalo (2009) and Pyrenean Ibex (2009).
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; human clones in the form of identical twins are common place, with their cloning occurring during the natural process of reproduction.
There are two commonly discussed types of human cloning; therapeutic cloning and reproductive cloning. Therapeutic cloning involves cloning adult cells for use in medicine and is an active area of research. Reproductive cloning would involve making cloned humans.
A third type of cloning called replacement cloning is a theoretical possibility combination between therapeutic. Replacement cloning would entail the replacement of an extensively damaged, failed or failing body through cloning followed by whole or partial brain transplant.
Ethical Issues of Cloning
Because of recent technological advancements, the cloning of animals (and potentially humans) has been an issue. Many religious organizations oppose all forms of cloning, on the grounds that life begins at conception, Judaism does not equate life with conception and, though some question the wisdom of cloning, Orthodox Judaism rabbis generally find no firm reason in Jewish laws and ethics to object to cloning.
From the standpoint of classical liberalism, concerns also exist regarding the protection of the identity of the individual and the right to protect one”s genetic identity.
Some people may be more open to the idea of cloning of animals because most western countries have passed legislation against cloning humans, yet only a few countries passed legislation against cloning animals.
Possible Abnormalities Due To Cloning
Researchers have found several abnormalities in cloned organisms, particularly in mice. The cloned organism may be born normal and resemble its non-cloned counterpart, but majority of the time will express changes in its genome later on in life.
The concern with cloning humans is that the changes in genomes may not only result in changes in appearance, but in psychological and personality changes as well.
The theory behind this is that the biological blue print of the genes is the same in cloned animals as it is in normal ones, but they are read and expressed incorrectly. Results of these abnormally expressed genes in the cloned mice were premature death, pneumonia, liver failure and obesity.
Cloning Extinct and End Angered Species
Cloning or more precisely, the reconstruction of functional DNA from extinct species has, for decades been a dream of some scientists. Vertebrate cloning poses little risk to the environment, but it can consume scarce conservation resources, and its chances of success in preserving species seem poor.
To date, the conservation benefits of transgenics and vertebrate cloning remain entirely theoretical, but many of the risks are known and documented.
Conservation biologists should devote their research and energies to the established methods of conservation, none of which require transgenics or vertebrate cloning.
In summary, in this article, cloning, which in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually, was discussed.
There are molecular cloning, cell cloning, cloning stem cells, organism cloning, horticultural cloning and parthenogenesis. Of priority interest to us is the species cloning. Many species have been cloned, but there are ethical issues regarding the cloning that cannot be ignored.
Glossary
Abdominal Ballottement – This involves palpation to determine pregnancy by pushing up against the uterus with the finger so as to feel any embryo as it sinks back in to place in the amniotic fluid.
Artificial Insemination (AI) – The possible impregnation of a female by artificial introduction of semen taken from a male.
Cloning – The process used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms. It is the process of producing similar populations of genetically identical individuals.
Oestrus (Heat) – The time within the oestrous cycle when the female animal stands to be mounted and mated by the male.
Cryopreservation – A process where cells or whole tissues are preserved by cooling to sub – zero temperature typically – 196oC.
DNA Probing – The binding of an agent directly to a predefined sequence of nucleic acids.
Embryo Transfer (ET) – The technique of recovering embryos from the reproductive tract of the donor animal and then inserting same into the reproductive tract of the recipient or foster mother.
Non Return to Oestrus – When an animal is mated or inseminated and it does not return to oestrus.
Nuclear Transfer– This is a form of cloning.
Oestrous Cycle – The cycle from one period of heat to the other in the same female animal.
Sex – The sum total of those differences in structure and function on the basis of which an organism is classified as male or female.
Super Ovulation – Used when in response to some treatment, the number of follicles ovulating is significantly above the normal ovulation rate for the species or breed in question.
Synchronization of Oestrus – a term used to indicate the process of bringing groups of animals into heat together in response to some form of treatment.
Time of Ovulation– The moment of follicular collapse with release of egg(s).
Weaning– The process of causing the young to become accustomed gradually to food other than its mother’s milk.
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