Molecular, Cell and Organism Cloning Techniques

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Table of Contents

Introduction
The Technique of Cloning
Conclusion
References

Introduction

Cloning is the process of creating a physical entity that is a precise copy of another organism or cell. The origin of the word “clone” is no linked to genetics. This term was created in 1903 by Herbert J. Webber, a plant physiologist, who used a Greek word klon to identify the plants received by gardeners after budding, bulbing, or cutting.

Cloning can be either natural or artificial. Natural cloning can be seen in the process of cell replication. Organisms with no cell nucleus (prokaryotic organisms) demonstrate cloning processes as well; for instance, yeasts and bacteria use budding or binary function to create identical versions of themselves. Eukaryotes (organisms will a present cell nucleus) have cells that reproduce by the means of mitosis, which is dividing itself to produce a clone. Multicellular organisms can undergo a natural cloning as well. It can occur in different ways. For example, asexual reproduction is a natural way of cloning, which lies in reproduction without fertilization.

It is a way of reproduction for many plans: a separated part of a plant grows into a new plant. In agriculture, people use this opportunity to expand the quantity of crops by increasing the number of necessary plants; it is not considered artificial cloning since they use the natural faculties of these plants and do not make any genetic changes. Some insects also reproduce in such away. In the natural world, such a phenomenon as parthenogenesis exists. Parthenogenesis is a self-reproduction of a multicellular organism without a mate and, hence, without fertilization. The examples of species, who commonly use parthenogenesis, can be found among insects, nematodes, crustaceans, and even fish.

Aside from natural cloning, artificial ways of cloning exist. In biology, cloning is understood as a duplication of biological material – DNA, a cell, or a whole organism. For these three types of material, different techniques were developed by scientists. Artificial cloning faces a strong ethical controversy in the society. Many people consider that artificial cloning is a breach of natural laws, even though cloning exists in a natural version. Artificial cloning is used for research, as well as for medical and reproductive purposes (Rugnetta, 2015).

The Technique of Cloning

Molecular Cloning

Molecular cloning, also known as gene cloning, is the copying of a DNA fragment or sequence. It is used for experimental purposes, as well as for practical tasks, such as DNA profiling, which allows finding a person with the use of their fingertips. The necessary conditions to enable an act of molecular cloning are the following:

for the supplementation of a DNA sequence to an organism, it is needed to have that sequence connected to the origin of replication, i.e. that very point in DNA, where the process of replication is started;
the performers of cloning should have special cloning, which are fragments of any organism’s DNA that can be successfully amplified in another organism.

DNA cloning commonly involves four main steps: breaking one DNA strand into pieces, constructing a needed sequence out of those pieces, injecting this sequence into a cell, and, finally, selecting the cells that have successfully undergone this process. To make the cell accept the inserted DNA fragment, the influence of a DNA enzyme, light, chemicals, gene gun, and electrical fields can be used. A typical DNA cloning process is done through the following stages: selecting a host organism and the source of a cloning vector, preparing the DNA of the mentioned source, preparing the DNA for the cloning process, generating a laboratory DNA, inserting the latter DNA into the host organism, choosing organisms that contain generated DNA, and selecting the clones that have successfully acquired the needed properties (Green & Sambrook, 2012; Brown, 2010).

Cell Cloning

Cell cloning can be defined as the cloning of a unicellular organism, for instance, a bacteria. In such case, three cloning techniques exist dilution, which needs a suspension of cells with certain concentration to be prepared; cloning ring technique, when cells are planted into special cell culture dishes; robotic transfer system, when the whole process is conducted by a special computer platform, which performs the transition of cells from a dish to a plate with growth medium (McFarland, 2000).

More often, the term “cell cloning” is used to define the cloning of stem cells, also known as somatic-cell nuclear transfer (SCNT). SCNT is used for medical and research purposes. The cloning of stem cells allows to cure human diseases more effectively, but there is still a need for further research, which will discover more opportunities for the medical use of cell cloning. The technique of SCNT lies in the following. The oocyte of the host organism is caught at metaphase II and stopped by the means of light distributed through a pipette.

Then, a small fragment of the zona pellucida is removed with a glass needle and inserted through this piercing to take out the nuclei of the oocyte and the polar body. Next, the somatic nuclei need to be introduced into the enucleated oocyte. This can be done through electrofusion or by placing the somatic nucleus to the perivitelline space. After that, growth hormones and specific markers are added to enable the introduction of the cloned cells to an organism (Kfoury, 2007).

Cloning an Organism

Cloning a living multicellular organism is usually defined as reproductive cloning. During this process, an identical copy of an existing organism is received. In theory, humans can successfully undergo cloning, but a lot of contradiction, primarily ethical and social, arising from such a possibility, which hinders the research process, sometimes in a legal way. For that reason, reproductive cloning is tried only on animals. An animal, which appears as a result of reproductive cloning, has DNA identical to the initial animal. One of the most famous examples is Dolly the sheep.

The most widely known technique for reproductive cloning is the use of somatic cell nuclear transfer when the genetic material of a nucleus from a donor’s body cell is transported to the “empty” egg, whose genetic material was removed with its whole nucleus. After that, the egg with the new DNA is exposed to the influence of electric currents or chemicals to trigger the division of cells. Then, the embryo inside the cell develops, and once it reaches a certain stage, it should be put into the uterus of a female specimen to provide it with favorable conditions until the fetus is fully developed. The born organism will be the clone.

However, it is not an identical copy of the donor in a full sense; Dolly is no exception. Such clones indeed receive the nuclear or chromosomal DNA of the donor, but some of the genetic materials of the clone originate from the mitochondria, the power sources of the cell, which is located in the cytoplasm of the egg that was enucleated. Mitochondria possess their DNA. The mutations that occur in mitochondrial DNA must have a serious impact on the aging process (Biological and Environmental Research Information System, 2009).

The example of Dolly proved that it is possible to reprogram the genetic material taken from an adult cell to produce only such genes as are necessary for udder cells, which enables to create a whole new organism. This example is important because before Dolly scientists considered that there was no way to make a cell of one type out of a cell of another type; in other words, they believed that a liver cell could not be transformed into a heart cell, and vice versa. Now science knows that such transformations are possible. However, the tools of humanity are still imperfect. The reprogramming process is far from ideal, which leads to errors that cause early death and diseases in animal clones (Biological and Environmental Research Information System, 2009).

The use of induced pluripotent stem cells (iPS cells) by the means of reprogramming is a rather new technology employed for reproductive cloning.

An unusual method of artificial organism cloning is the twinning, or splitting, of an embryo. In this process, an initial embryo is divided into two separate embryos. Then, these two embryos are placed into the uterus of a female specimen (or a female human). Splitting is done by expanding an external fertilization of an egg on the 6-cell to 8-cell stages. In the case of success, the female gives birth to monozygotic twins, who are basically clones (Illmensee, Levanduski, Vidali, Husami & Goudas, 2010).

Except for the twinning method, it is not possible to speak about human cloning yet. Some scientists are opposed to cloning humans, and not only for ethical reasons: only 1-2 cases out of 100 are successful in animal cloning, and many scientists are not ready to put human lives at risk. Additionally, around 30% of animal clones suffer from “large-offspring” syndrome and other problematic conditions. Some animal clones die young due to the complications after cloning and infections.

Human clones will probably have the same problems. Moreover, the impact of cloning on mental development is unknown; animals do not have self-reflection and self-esteem, but humans do, and being a clone can have a dramatic negative effect on mental health. Thus, a double problem arises: on the one hand, more experiments are needed to advance the technology of cloning, but, on the other hand, further experimentations are hindered by ethical, social, and psychological concerns (Biological and Environmental Research Information System, 2009).

Conclusion

The term “cloning” means creating an alive object that is a replication of another alive object. Cloning exists as a natural phenomenon. It can be found in the process of cell reproduction. Both prokaryotes and eukaryotes can undergo cloning as a means of reproduction. Plants can reproduce by division. Multicellular organisms also reproduce by parthenogenesis.

Artificial cloning includes molecular and cell cloning and cloning an organism. Molecular cloning, also gene or DNA cloning, involves copying a fragment of DNA of one cell and introducing it into another cell. Cell cloning include the cloning of unicellular organisms, as well as somatic-cell nuclear transfer. Cloning an organism is quite similar to cell cloning, but genes are introduced into an egg, which needs a female specimen to bear it.

References

Biological and Environmental Research Information System. (2009). Cloning fact sheet. Web.

Brown, T.A. (2010). Gene cloning and DNA analysis (6th ed.). Chichester, UK: John Wiley & Sons.

Green, M.R. & Sambrook, J. (2012). Molecular cloning: A laboratory manual (4th ed.). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.

Illmensee, K., Levanduski, M., Vidali, A., Husami, N.& Goudas, V.T. (2010) Human embryo twinning with applications in reproductive medicine. Fertility and Sterility, 93(2), 423-427.

Kfoury, C. (2007). Therapeutic cloning: Promises and issues. McGill Journal of Medicine , 10(2), 112-120.

McFarland, D. (2000). Preparation of pure cell cultures by cloning. Methods in Cell Science, 22(1), 63-66.

Rugnetta, M. (2015). Cloning (Genetics). In Encyclopaedia Britannica. Web.

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