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Cloning: Simple explanation through examples
#1
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When you think of cloning, Dolly the sheep is probably the first thing that comes to your mind. Artificial cloning came later; we stole that idea from nature. Asexual reproduction, typical for so many animals, is natural way to reproduce. New individual is created by division of the mother cell giving daughter cell with the same genetic material. Biotechnology found a way to utilize this natural process for production of novel molecules, cells or even organisms. Main purpose is to help solve certain medical issues or reveal genetic mysteries in various experiments that are taking place all over the world.

Molecular cloning is used to amplify DNA sequence (gene, promoter, non-coding sequence…) of interest. To ensure replication, DNA sequence must be linked with origin of replication – part of DNA that will initiate replication. Ligation is process of inserting DNA sequence into cloning vector (peace of DNA, carrier of the sequence). DNA ligaze will connect sequence and vector by “gluing” sticky ends at each DNA piece. Transfection of cell with vector carrying sequence of interest is next step. Electroporation, optical injection or biolistics are mostly used transfection techniques, but they are not successful always. Additional genes in cloning vector are necessary to ensure easy recognition of cells containing DNA piece of interest. Some of the most famous “markers” used are genes providing antibiotic resistance (when substrate with antibiotic is used for cell growing) or color markers (for blue/white cell screening). After cell colonies are formed, DNA sequence will be multiplied and analyzed using PCR, DNA sequencing or restriction fragment analysis.

Cellular cloning is production of cells containing same genetic info as mother cell. Cells derived from multi-cellular organism are much more complicated to clone than cells that are unicellular by nature. Technique “clone rings” is used for cloning multi-cellular organism derived cells. Cell suspension is exposed to mutagenic agent or drug and planted at high dilution, which result in new colonies formation. On a stage of few cells created, trypsin and polystyrene rings (covered in grease) are placed over each formed colony. Cells from the inner part of the ring are collected and moved to another substrate to develop further. Cellular cloning could solve serious medical issues that are non-treatable by conventional medication (such as Alzheimer disease). Cells used for this purpose are stem cells - as they could give raise to any cell lineage we want. SCNT (Stem Cell Nuclear Transport) is used for developing embryonic stem cells (ESC) that will have both research and therapeutic application. ESC are created by removing nucleus from the egg and implanting nucleus from adult somatic cell (containing both mother and father genetic material). Egg will act like it’s been fertilized and start dividing first to reach blastocyst stage and then toward any cell lineage we want. Procedure is the same with animal species and could be used to produce additional food source (by cloning farm animals) or to prevent extinction of endangered animals. It may sound like simple process, but success rate with this kind of genetic manipulation is pretty low. Dolly the sheep was first mammal created in laboratory. Out of 277 eggs used for SCNT, just 29 embryos were created. 3 survived until birth and only one - more famous by its given name – Dolly, survived until adulthood. Although, genetic material in the newly formed cell (organism) is the same as in donor’s cell, certain part of DNA is unique. Each cell contains mitochondria with its own genetic material. It’s inherited solely from the mother due to couple of reasons: egg contains more mtDNA than sperm; sperm derived mtDNA is easily degraded once inside or it can even fail to enter the egg. Thanks to this phenomenon, cloned cells can be considered genetic hybrids, as they contain both somatic DNA and mother mitochondrial genes.

Some large animals can create clones on their own. Lizards, snakes, ants, crustaceous species and even certain sharks are able to produce new individual by parthenogenesis – out of unfertilized egg. For most species, this is not obligatory way to reproduce but a method to overcome crisis in their environment. Komodo dragon, for example, can reproduce by parthenogenesis to increase the population in the habitat and then switch back to sexual reproduction to increase genetic diversity of the next generation.

To conclude, cloning is not something we invented, it’s natural phenomenon that we start exploiting recently. Wise and careful approach could be beneficial for the planet; we just need to pay attention not to cross the line, as genetic diversity is what allowed us to survive so far.
#2
Besides technological, ethical and diversity issues, cloning still has numerous possible applications in our lives. Among them, medical applications are of course the most important and here are some of those:

Preclinical studies represent a necessary first step during the investigation of new medications. For the purposes of preclinical studies, laboratory animals are almost always used. Choosing the optimal experimental animals which contain properties we need for the research is not always an easy task, and cloning technology could provide a great help when it comes to this issue. Namely, we can choose one animal which has certain qualities we require, and then clone it many times thus getting a huge population of organisms with exactly the same genetic profile. That would make the results of our research more accurate and trustworthy.

Stem cells are pluripotent cells which are able to reproduce and differentiate into any human cell, and they can also be cloned for the purposes of research. In adults, the number of pluripotent stem cells is low, and they can only be extracted from certain areas of the body, so cloning those cells would enable more research in order to discover their abilities and behavior.

A very interested topic is also cloning the extinct species. In order to clone any organism, a preserved DNA molecule from at least one cell is needed. Tissue samples would probably be possible to acquire for some lately extinct species, but hardly for those which disappeared long time ago, such as dinosaurs. The more realistic and hopefully achievable option is the use of cloning techniques in order to protect some critically endangered species such as Amur Leopard (30 left), Sumatran Tiger (less than 400 left), and Mountain Gorilla (880 left). It is left to hope that cloning technology would be perfected soon enough to protect these organisms from extinction and help them repopulate.
Sasa Milosevic
  

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