Category: Cloning

Over the last few years, the science of reproductive cloning has sparked ethical debates. Though most fears associated with reproductive cloning are valid and significant, there are certain misconceptions that have led to unnecessary fear and trepidation. The most significant arguments against reproductive cloning are that it is wrong to make a copy of someone as it affects the uniqueness of the clone, violates the autonomy of the cloned individual, it is unnatural, affects human dignity, causes identity and psychological crisis in the cloned individual and also affects the cloned individuals from having an open future. While some critics agree to these arguments, a few consider these arguments to be mere assumptions and hypothetical. One of the major arguments in human reproductive cloning is the “life in the shadow” argument which claims that the cloned individual could be subjected to psychological distress and harm as a result of social pressures as the clone’s life would not be completely his or her own (1).The right to autonomy of the clone could be compromised as he could be given traits or characteristics that he or she would not find desirable. In this essay, I will discuss the ethical and moral considerations in the “life in the shadow” argument in human reproductive cloning by examining in detail the strong and weak points made in the claim. I will further explain my disagreement with the argument as I believe that despite having several strong points the argument is mostly based on assumptions, misinterpretations and is hypothetical concluding that, with claims against cloning based on life in the shadow argument cloning is still a viable option.

Firstly, the important debate in the reproductive cloning and in life in the shadow argument is that the right to autonomy of the clone would be largely jeopardized (2). It emphasizes that genetic individuality is an essential part of life as it provides an individual with an identity and makes him or her unique. Theorists like Tannert (3) and Holm suggest that people believe in genetic essentialism (relating the genotype to the physical characters and personality) and develop false hopes and expectations (4), the life of the clone is overshadowed by the genetic donor and the clone would not be free to make his or her own decisions in life. The accomplishments of the clone would also be always compared to its donor preventing the clone from leading an autonomous life. In my opinion, the genetic identity of the clone would be altered, which is against the natural way of things and the clone could grow up to find the science of cloning to be undesirable and autonomy compromising. In the case of a savior clone, the life of the clone would be used as means against his or her own consent. This would be an even more serious violation of the clone’s basic moral rights and his or her life would be completely overshadowed by the donor. The right to autonomy claims of the debate leads us to open future argument. This argument claims that the clone would lead a life which would merely be a partial re-enactment of the donor’s life forestalling its opportunity to an open future (5). Reproductive cloning could cause an infringement of the cloned individual’s right to ignorance as he or she would know too much about themselves. Buchanan et al states that:

“The idea is that parents have a responsibility to help their children during their growth to adulthood to develop capacities for practical judgment and autonomous choice, and to develop as well at least a reasonable range of the skills and capacities necessary to provide them the choice of a reasonable array of different life plans available to members of their society. In this view, it would be wrong for parents to close off most opportunities that would otherwise be available to their children in order to impose their own particular conception of the good life.”

He suggests that knowledge of the genetic identity could enable parents to restrict the exposure of children to develop the competence and capacity to choose their path in life (6). The genetic intervention in the cloned individuals could restrict their range of choices and options in life. The enhancement of particular genes could lead to the incapability of another trait which the cloned individual might have been interested in. The right to open future of the cloned individuals could be deeply affected as they have would have constrained and restricted environmental and genetic choices.

One of the major concerns in reproductive cloning is the wellbeing of the clone. Critics and bioethicists claim that cloning could cause deficits in the wellbeing of the cloned child than a child conceived by natural means. The CEJA notes suggest that cloning could cause psychosocial distress in the cloned individuals (7). The genetic identity of the clone provides insights on the potential of the cloned individual which increases the expectations and societal pressures on the clone as compared to a child born out of natural procreation. Dinc suggests that the knowledge of genetic identity could cause a great deal of harm to the clone such that the knowledge of being a cloned individual could invoke several emotional reactions and outrages. He or she could go through denial, identity crisis and show anger towards the parents leading to low self esteem. Conversely, knowledge about his or her genetic favorability could invoke a sense of superiority and over-confidence in the cloned individual affecting his or her mental health and well being (8).Genetic essentialism could affect the well being of the cloned individual as parents could have false beliefs on the competence of the child causing harmful implications on his or her well being wellbeing (4). If the clone is a savior clone, then his or her wellbeing is a major concern. Apart from leading a life under the shadow of the donor the care they receive from their parents is also greatly influenced by the donor. However there are several ethical concerns in the concept of savior clones which are not discussed as it is not within the scope of this essay.

The major pitfalls in the arguments in favor of the life in the shadow arguments is that most arguments points to genetic essentialism and they stand correct only if this experiential foundation remains true. The concept of genetic essentialism is based on the factual errors in people’s understanding of the genetic disposition and all arguments could fail if this misinterpretation is cleared. Though the right to autonomy of the clone individual is a major concern, Burley et al claims that the concept of autonomy is used by liberal theorists to defend a set of ways which they believe to bring meaning to life. They also suggest a flaw in assessing the degree of harm caused due to compromising the autonomy of the cloned individual to the harms caused to children raised from other acts of procreation which can cause similar or even severe consequences affecting the child’s autonomy (9). They claim that:

“We maintain that unless it is shown convincingly that ‘living in the shadow’ is somehow both horrendous and more autonomy-compromising than the plethora of other widely accepted and permitted upbringings a child might be ‘forced’ to undergo, the liberal principle of freedom in matters relating to procreation overrides the concern about cloning and child welfare autonomy-related welfare deficits that will be suffered by clones.”

They find the arguments against cloning pertaining to the autonomy and welfare of the cloned individuals to be unconvincing as children from other means of procreation could also be subjected to such implications. In my opinion, the right to autonomy and open future is also compromised in children living in conservative households wherein they are sometimes forced to follow the way of life of their ancestors. Though some ethicists believe in the open future argument and consider it wrong to violate the clone’s right to ignorance, as Kuhse suggests:

“While a cloned child may not have some of the opportunities of a non-clone, her future would be sufficiently open to allow her to live a life that is of benefit to her in a relevant sense. In what sense, then would she have been harmed by having been brought into existence?”

The cloned child would have several opportunities to thrive in their strong domains when compared to children born naturally who do not have the opportunity to identify or work on their strengths. The knowledge of genetic identity could also be beneficial than harmful. People nowadays voluntarily undergo genetic testing to identify diseases and obtain other relevant information. Kuhse questions the need to assume that this information would be appalling and cause harm rather than being helpful (5). Burley et al suggests that if exposure to information about genetic origin could traumatize and harm a cloned child the same is bound to happen to any child born by other means of procreation (9). I believe, that children could gain knowledge about their parents and ancestors past and would face the same circumstances as the clone, be it good or bad. If genetic origin shadows the life of a cloned child, the similar could happen to any child born by any means of procreation.

In conclusion, though the life in the shadow argument has several strong points and aims to protect the dignity and life of the cloned individuals it fails to assess the extent of damage it causes in comparison to children born naturally or by other means of procreation. The wellbeing of the cloned child is of principle importance and it is the responsibility of the society to provide the best to our future children. Assuming the worst out of the situation and arguing that the welfare of the cloned child would be adversely affected is wrong. On the contrary, it is also possible to assume that the cloned child would be loved, cherished and be provided the freedom to choose its own path. The cloned child could also have several advantages and be provided with a better future. The right to ignorance of the cloned child can be protected by withholding critical information from the child till he or she is autonomous. Though the autonomy of the cloned child could be affected, there is a possibility he or she could have a better life despite not being able to exercise his right to autonomy. Therefore, I believe that, while there are several ethical and moral concerns pertaining to the life in the shadow argument of reproductive cloning, anticipating bad outcomes and prohibiting cloning on the grounds of this argument is prejudiced and must be assessed in a manner that brings about maximum benefits to mankind.

The cloning process is taking cells from an individual and replicating genes or DNA. In the process of cloning the gene is entered in the plasmid, which is a piece of the DNA (Overview: DNA cloning, 2019). There are four steps into the cloning process (Eric J. Simon, 2016). The steps are as follow;

• Put the gene in the plasmid and restricting the enzymes
• Add plasmid into bacteria
• Make more protein, harvest and purify protein (protein production)
• Uses of DNA cloning

The first step is by restricting the enzymes, which causes DNA ends to have short and stranded overhangs (Overview: DNA cloning, 2019). If overhangs match, they will form a pair and stick together (Overview: DNA cloning, 2019). Then the second process would be adding plasmid into bacteria, which consists of heat shock to bacterial cells which encourages the cells to take foreign DNA (Overview: DNA cloning, 2019). Bacteria without plasmid will not survive and will die (Overview: DNA cloning, 2019). The third process is protein production where plasmid-bearing bacteria triggers chemicals acts as a protein factory and purify them into producing large amounts of protein (Overview: DNA cloning, 2019). Finally, the uses of DNA cloning, which is used as a multipurpose in molecular biology such as;

• Biopharmaceuticals
• Gene therapy
• Gene analysis (Overview: DNA cloning, 2019).

There are advantages and disadvantages of cloning, the advantages in human gene therapy (Overview: DNA cloning, 2019). Human gene therapy is where diseases are treated by providing new genes, which replaces or supplements the defective gene (Eric J. Simon, 2016). Once disease that cured around 22 children between the years of 2000-2011, was severe combined immunodeficiency (Eric J. Simon, 2016). These patients would have ended up dying from this disease without treatment (Eric J. Simon, 2016). Another advantage, which is like treatment of a disease, but it increases life expectancy for the human race (Ayres, 2019) The disadvantage of cloning could be the loss of diversity of genes and could cause an imbalance to society (Ayres, 2019).

Cloning and genetically modified organisms are two ways biology is used to produce organisms that are needed (Lanka, 2018). Cloning is a creation of cells which are genetically identical to the originals (Lanka, 2018). Cloning maintains characteristics of organisms over time (Lanka, 2018). Genetic engineering is an alteration in DNA to be able to create new organisms by adding or taking away genes (Lanka, 2018). The similarities that they both have are they create valuable organisms, production of hormones and pharmaceutical products (Lanka, 2018). Plants, food and drug production is the most commonly used production of genetically modified organisms (Theresa Phillips, 2008). One of the most highlighted dangers would be exposure to new allergens and antibiotic resistance gene’s (Theresa Phillips, 2008). This causes a danger because humans become allergic to GMO foods. Then when dealing with antibiotic resistance once they body catches an infection, they are unable to fight the infections naturally and when given antibiotics the body fights the medication which causes the infection to continue within the body.

Gene therapy as mentioned before at the beginning gene therapy is very helpful to those 22 children with severe combined immunodeficiency. Gene therapy can help those with non-life-threatening illnesses to be able to get relief and possibly fertility issues. Gene therapy has promising outcome for our future, however right now there is little evidence and little research done so far (Eric J. Simon, 2016). It provides those with no hope at a future and now they are planning their future.

References

1. Ayres, C. (2019). 11 Advantages and Disadvantages of Cloning. Retrieved from Vittana: https://vittana.org/11-advantages-and-disadvantages-of-cloning
2. Eric J. Simon, J. L. (2016). Campbell Essential Biology with physiology 5th edition. Pearson Learning.
3. Lanka. (2018, 02 26). Difference Between Cloning and Genetic Engineering. Retrieved from PEDIAA: https://pediaa.com/difference-between-cloning-and-genetic-engineering/
4. Overview: DNA cloning. (2019). Retrieved from Khan Academy: https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloning-tutorial/a/overview-dna-cloning
5. Theresa Phillips, P. (2008). Genetically Modified Organisms (GMOs): Transgenic Crops and Recombinant DNA Technology. Retrieved from Scitable: https://www.nature.com/scitable/topicpage/genetically-modified-organisms-gmos-transgenic-crops-and-732

A gene is a nucleotide sequence which dictates the synthesis of a particular RNA or protein molecule. Their control over the produced proteins govern both phenotypical and genetic traits, including susceptibility to diseases like Cystic Fibrosis. Driving gene expression is Central Dogma, a two-step process in which DNA is converted to an intermediate RNA (mRNA) through transcription, then from mRNA to protein through translation. Virtually all living and acellular beings abide to Central Dogma bar the Retrovirus family and viruses such as Hepatitis B Virus (HBV) (Madigan et al., 2012). These specialised viruses are capable of producing an enzyme called Reverse-Transcriptase (RT) and this converts their single-stranded RNA molecules into double-stranded complementary DNA (cDNA), allowing them to integrate into the host’s genome in a process termed lysogeny (Madigan et al., 2012).

As technology and the understanding of genomics thrive, diseases once thought incurable are being corrected through a revolutionary process called Gene Therapy. Gene therapy is where a gene is delivered to an individual in order to correct a genetic disease caused by a faulty version of the replacement gene. There are two versions of gene therapy, somatic and germ-line. Somatic is limited to the lifetime of the individual while germ-line prevents the defective gene from being passed onto future descendants, the latter being considered highly unethical and currently banned due to its ability of affecting subsequent generations. To generate new and functional copies of defective genes, DNA cloning vectors are essential. A vector is a virus or DNA which can be utilised to carry and replicate other pieces of DNA. (Thieman and Palladino, 2009). Ever since the construction of pSC101, the number of DNA vectors has exponentially increased with plasmids being the most popular cloning vectors for being easily transformable and isolatable from their host bacterium. Unfortunately, plasmids have their limitations just like all the other vectors in Table 1. Thus, to undergo Gene therapy, it requires several vectors (Thieman and Palladino, 2009).

Previously, genes for deficiencies such as insulin deficiency in diabetes mellitus patients were taken from animals, but this carried risk of zoonotic diseases. Thus, the understanding of bacterial genomics was ground-breaking. Certain species of bacteria are competent, meaning that they are willingly able to uptake naked DNA strands which shall give them a selective advantage (Madigan et al., 2012). Escherichia coli (E. coli) is the most studied model bacterium and is favoured for majority of lab work, particularly in recombinant DNA cloning practises. For transformation to occur, the plasmid DNA vector needs to contain several characteristics, including a Multiple Cloning Site (MCS), Antibacterial resistance gene, Origin of replication (Ori) and selectable marker genes.

The antibacterial resistance gene is highly important as it indicates which colonies have been transformed and house the target gene. For example, if the growth medium was embedded with Ampicillin, the plasmid DNA would be constructed to contain the AMPR gene so that only competent bacterium cells would be present on the agar plates (Thieman and Palladino, 2009). The colonies are grown until stationary phase (optimum stage) and one colony is selected and grown in a selective liquid culture medium. The grown E. coli cells are centrifuged into a pellet which is subjected to the PureLink Quick Plasmid Miniprep protocol. The first step is resuspension. Resuspension buffer uses EDTA, a chemical which chelates the divalent cations Mg2+ and Ca2+, Dnase cofactors that degrade plasmid DNA. However, with EDTA present, the cofactors cannot initiate Dnase activity and thereby protects the plasmid DNA. Rnase is also present and gives a purer DNA sample by degrading RNA present in the cell. Next, lysis buffer, which contains Sodium Hydroxide (NaOH) and Sodium Dodecyl Sulphate (SDS), is added. NaOH denatures any proteins and SDS lyses the cells’ membranes. Potassium acetate, an acidic salt, is added to neutralise the basic effect of NaOH and the degraded proteins shall precipitate and are easily removed by centrifugation, leaving the supernatant containing the plasmid. The supernatant is transferred to a spin column where the plasmid DNA binds. Any impurities present are washed away using an ethanol wash, leaving the plasmid DNA bound to column while the flow-through accumulates in the wash column before being discarded.

After two centrifugations, the column is placed into a recovery tube and centrifuged so that the plasmid DNA is eluted by Tris-HCl from the column into the recovery tube. It’s critical that the recovered plasmid DNA is moderately concentrated and pure as otherwise the remaining stages are affected. Both of these qualities are tested by use of gel electrophoresis. By using gel electrophoresis, the DNA bands which appear shall not only indicate the band size but also its purity as, the brighter it is, the purer it is. If no band appears, it indicates that the bacterium wasn’t competent or that the protocol aforementioned wasn’t strictly followed. To quantify the results, one could use spectrophotometry or fluorometry to determine the concentration of plasmid DNA. The next stage involves restriction digestion. This stage is crucial because, to ensure efficient transformation into the destination vector, specific restriction enzymes are chosen to act as surgical scissors, cutting in-between specific nucleotides in the DNA sequence and generating “sticky ends”. The term “sticky ends” refers to both ends having an uneven distribution of nucleotides on either end. The samples are then loaded onto a gel electrophoresis with the purpose being to calculate how much insert needs to be added during ligation. The bands are viewed under UV light, photographed and cut out quickly to avoid risk of thymine-dimerization. The gel slices are dissolved and purified, and the contents mixed together, allowing the linearized vector to ligate into the destination vector which has had an exact same cut in its own DNA sequence, thereby ensuring that the donor vector’s DNA adherently binds in the correct conformational orientation.

Once ligated, the same transformation process from earlier is used. Only transformed bacteria with the destination vector will grow in colonies on the plates. To confirm the ligation was successful, random colonies will be selected for Polymerase Chain Reaction (PCR).

(1) Double-stranded DNA is denatured at 950C, leaving single-stranded DNA molecules. (2) Temperature is lowered between 40-600C depending on primers. The primers anneal to the strands, flanking the site of interest. (3) The temperature is raised to 720C where extension occurs. Taq DNA Polymerase synthesises new DNA strands, creating two new double-stranded DNA molecules. The steps repeat for another 30 cycles with the number of target DNA being generated exponentially increasing.

(1) Double-stranded DNA is denatured at 950C, leaving single-stranded DNA molecules. (2) Temperature is lowered between 40-600C depending on primers. The primers anneal to the strands, flanking the site of interest. (3) The temperature is raised to 720C where extension occurs. Taq DNA Polymerase synthesises new DNA strands, creating two new double-stranded DNA molecules. The steps repeat for another 30 cycles with the number of target DNA being generated exponentially increasing end product being ran through gel electrophoresis and scanned with a specialised probe.

Today, gene therapy is necessary to treat genetic disorders, including dominant and recessive genetic disorders. Table 2 showcases several medical conditions currently being studied and at what trial stage. Gene therapy can take one of two approaches; viral and non-viral. Viral delivery is more specific to disorders affecting the central nervous system (CNS) as the majority of other available therapeutic approaches struggle to breach the blood-brain barrier and those that do fail to have effects on the target cells (Qu et al., 2019). As for non-viral delivery, methods include using a Gene-gun, a direct contact methodology in which a high-velocity injection of DNA-coated fragments transform the cells, direct injection of the vector, and liposomes, small spheres that contain DNA with artificial membranes which can fuse with target cells’ membranes and release their contents (Krebs et al., 2014). Liposomes theoretically fuse with any cell type, but their affect is only short-lived, and symptoms start to reappear. Direct injection of the vector has proven to be a worthwhile methodology, especially in the treatment of lung cancer. By mixing a functional p53 gene into an injectable drug, the gene can bypass the immune system reaction, a frequently occurrent problem with other gene therapy techniques, and suppress cell tumour growth (Borem et al., 2003).

Viral vectors however are more promising than non-viral delivery methods. Table 3 showcases several viral vectors currently in use. After much development, advancement towards the creation of innovative viral vectors with immunogenicity delivery with greater efficiency and low genotoxicity is becoming a reality with adeno-associated virus (AAV) being at the forefront. AAV is believed to be a beneficial vector due to its distinct structures, allowing novel treatments of neurodegenerative diseases in the CNS (Qu et al, 2019).

Parkinson’s disease GDNF AAV2 Putaminal 25 NCT01621581 Phase I, active not recruiting Convection-enhanced delivery method was performed to infuse virus vectors into the brain

Vaccinia virus 25Kb Non-integrating Dividing and non-dividing cells Transient (only last short time) Huntington’s Disease (HD) is an autosomal dominant disorder which is passed from an affected parent to half their offspring, causing progressive degeneration/death of striatal neurons. Several studies have been conducted with one study highlighting that a SIRT3 gene, once delivered by an AAV vector, could prevent neurodegeneration from occurring within HD mice (Dufour et al., 2014). The delivery of SIRT3 prevented mitochondrial oxidative stress from occurring and thus maintained neuronal bioenergy. By 2018, laboratory and preliminary clinical research suggests that clinically applied treatment in regard to HD is now plausible (Qu et al., 2019).

Canavan disease is an autosomal recessive neurodegenerative disease which is caused by diffused spongiform white matter degeneration in the brain alongside dysmyelination and intramyelinic oedema. The condition arises due to aspartoacylase being inactive, thereby allowing Nacetylaspartate to toxically accumulate (Qu et al, 2019). Studies show that gene therapy would be beneficial to the patient, particularly when younger as, during a clinical trial in 2012, 13 Canavan disease patients received AAV2-aspartoacylase intraparenchymally with the long-term effects being recorded. All 13 patients responded well to treatment with no serious side effects developing (Ahmed and Gao., 2013). Therapeutically, the amount of Nacetylaspartate in the brain had decreased, seizures became less frequent and progressive brain atrophy was delayed. As a result, it’s recommended that early therapeutic intervention is taken to prevent the disease from escalating (Qu et al., 2019). Despite AAV being highly successful, there have been other trials in which the end results portrayed AAV as ineffective but, some of the trials potentially lacked a complete thorough knowledge about the disease itself. Clinically, AAV has other opposition, particularly neutralising antibodies. Any pre-exiting antibodies of AAV have caused an increasingly small limit of AAV vector usage in clinical gene therapy. (Ortolano et al., 2012; Rapti et al., 2012).

With gene therapy still a recently novel scientific field, it has a promising future despite its unfortunately morbid start. On September 18, 1999, Jesse Gelsinger, whom was ornithine transcarboamylase deficient, an enzyme essential for ammonia metabolism, died after the gene therapy he received caused him to undergo hepatic and respiratory failure. After a temporary moratorium on human clinical trials, the FDA allowed further human-related investigations to continue upon the advancement of Biotechnology practises such as vector construction for cloning and improvements in the transformation of competent cells by protocols like PURELINK (Borem et al., 2003). Abina et al. (2015) had a major breakthrough in gene therapy by identifying the genetic anomalies of the rare, congenital immune and platelet deficiency, X-linked Wiskott-Aldrich syndrome (WAS), therapeutically correcting it by injecting a lentiviral vector with the target WASp gene into the cells before being reinjected into the patients whom, at the time, had been undergoing chemotherapy so that the newly corrected stem cells would differentiate instead of their defective cells. After nine months, the six subjects’ immune systems were restored, and their clinical condition improved. With the scientific community captivated by the wonders of gene therapy and its recent successes within the last decade, particularly with viral-delivery vectors, it’s only a matter of time before all of the technical aspects and scientific details are developed and gene therapy becomes a staple in genetic disorder treatment.

References

1. Abina, S. H. B., Gaspar, B., Blondeau, J., Caccavelli, L., Charrier, S, Buckland, K., Picard, C., Six, E., Himoudi, N., Gilmour, K., et al. Outcomes Following Gene Therapy in Patients With Severe Wiskott-Aldrich Syndrome. JAMA, 2015; 313 (15): 1550 DOI 10.1001/jama.2015.3253
2. Ahmed, S. S., Gao, G. (2013). Gene therapy for Canavan’s disease takes a step forward. Mol Ther 21:505-506
3. Borem, A., Santos, F. R., and Bowen, D. E. (2003) Understanding Biotechnology. Pearson Education
4. Dufour, B. D., Smith, C. A., Clark, R. L., Walker, T. R., McBride, J. L. (2014) Intrajugular vein delivery of AAV9-RNAi prevents neuropathological changes and weight loss in Huntington’s disease mice. Mol Ther 22:797-810.
5. Krebs, J. E., Goldstein, E. S., and Kilpatrick, S. T. (2014) Lewin’s GENES XI. Jones and Bartlett Inc, Sudbury.
6. Madigan, M. T., Martinko, J. M., Stahl, D. A. and Clark, D. P. (2012). Brock Biology of Microorganisms, 13th Ed. Pearson Benjamin Cummings, San Francisco.
7. Nelson, D. L. and Cox, M. M. (2008). Lehninger Principles of Biochemistry, 5th Ed. W.H. Freeman and Co Ltd, New York.
8. Nesbit, A. 2017. BMS310 Applied Genetics: Genetic approaches to treating disease, online course materials, Semester 1, 2017, University of Ulster.
9. Ortolano, S., Spuch, C. and Navarro, C. (2012). Present and future of adeno associated virus based gene therapy approaches. Recent Pat Endocr Metab Immune Drug Discov 6: 47-66.
10. Pray, L. (2008) The Biotechnology Revolution: PCR and the Use of Reverse Transcriptase to Clone Expressed Genes. Nature Education 1(1):94
11. Rapti, K., Louis-Jeune, V., Kohlbrenner, E., Ishikawa, K., Ladage, D., Zolotukhin, S., Hajjar, R. J. and Weber, T. (2012). Neutralising antibodies against AAV serotypes 1, 2, 6, and 9 in sera of commonly used animal models. Mol Ther 20:73-83.
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13. Thieman, W. J. and Palladino, M. A. (2009). Introduction to Biotechnology, 2nd Ed. Pearson Benjamin Cummings, San Francisco.
14. Qu, Y., Liu, Y., Noor, A. F., Tran, J. and Li, R. Characteristics and advantages of adeno-associated virus vector-mediated gene therapy for neurodegenerative diseases. Neural Regen Res 2019;14;931-8

RECOMBINANT DNA TECHNOLOGY

Recombinant DNA Technology refers to the process by which DNA molecules of two different species are joined together and then inserted into a host for the production of new genetic combinations which are valuable to science, medicine, agriculture and industry.

Steps involved in this process are:

1. Isolation of genetic material

The genetic material in living organisms is generally nucleic acids. Whereas in most of the cases it is DNA and sometimes it is RNA. The first step in the process of RDT is to isolate the desired DNA in free form i.e. free from other macromolecules. So as DNA is bounded in the cell membrane along with several other molecules, enzymes are required to break all those barriers for its isolation which can be:

• a. Lysozyme
• b. Cellulase
• c. Ribonucease
• d. Protease

After the removal of the other unnecessary particles, addition of ethanol allows DNA to precipitate out as fine threads.

2. Restriction Enzyme Digestion

The restriction enzymes act as molecular scissors that cut DNA at specific sites. Agarose Gel Electrophoresis is performed in this process. Here, DNA is run on agarose gel and current is passed. In result, negatively charged DNA moves to positive electrode and settles according to size. This allows the separation and cut of digested DNA fragments.

3. Amplification using PCR

In PCR, multiple copies of DNA is made using enzyme DNA Polymerase. It helps to amplify few copies of DNA into millions of copies.

4. Ligation of DNA molecules

Purified DNA and vector of interest are cut using restriction enzymes. This gives the cut DNA and vector fragments. The process of joining these is Ligation with the enzyme DNA ligase. The resulting DNA is recombinant DNA.

5. Insertion of recombinant DNA into host

Transformation is the process by which recombinant DNA is introduced into a host. Also the bacterial cells should be treated to make them competent to accept new DNA.

6. Obtaining foreign gene product

New DNA multiplies inside the host and is expressed as protein. This is now a recombinant protein.

7. Downstream processing

Before the protein is released it is subjected to downstream processing that includes:

• a. Separation & Purification
• b. Formulation
• c. Clinical safety tests
• d. Quality control tests

RECOMBINANT DNA FORMATION

· DNA CLONING

DNA Cloning is an important process in Recombinant DNA Technology that has provided the scientists with the ability to produce many copies of a single fragmented DNA. This is done by inserting a small fragment of DNA into a DNA molecule which is allowed to replicate in a growing living cell such as a bacterium. The small replicating molecule is a DNA vector or carrier. The most commonly used vectors are plasmids, viruses and yeast cells.

Steps involved in this process:

1. Cutting and Pasting DNA

Restriction Enzymes are used for cutting DNA fragments.

Digestion is done

• To the plasmid which has a single cut site
• To target gene fragment which has a cut site near each end

DNA Ligase is used to combine fragments that allow them to link and make a recombinant plasmid containing the gene.

2. Bacterial Transformation and Selection

During transformation, specially prepared bacterial cells are given a shock that allows them to take up the foreign DNA.

After the bacteria get DNA they form colonies.

3. Protein Production

After the bacteria form colonies they are given a chemical signal that instructs them to produce target proteins. Once it forms, bacterial cells can be split open to release it. The proteins are then purified and can be used in various experiments.

Recombinant DNA Technology is useful in Industrial purposes in terms of commercial importance, improvement of fermentation processes and production of proteins from wastes.

· PROBLEMS WITH RECOMBINATION TECHNIQUE

Most of the cons of Recombinant DNA Technology are ethical in nature. According to some of the organizations, it is seen to have felt that this technology goes against the laws of natures or against the religious beliefs. Some worries are also been seen that if companies can allow scientist with patent to buy or sell genetic materials then it could become an expensive commodity. Problems with the safety of modified foods and medicines also occur.

REFERENCE

1. Brondyk, W. H. (2009). Chapter 11 Selecting an Appropriate Method for Expressing a Recombinant Protein. Methods in Enzymology. 463. pp. 131–147. doi:10.1016/S0076-6879(09)63011-1. ISBN 9780123745361. PMID 19892171.
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4. Johnson, I. S. (1983). ‘Human insulin from recombinant DNA technology’. Science. 219 (4585): 632–637. doi:10.1126/science.6337396. PMID 6337396.
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7. Watson, James D. (2007). Recombinant DNA: Genes and Genomes: A Short Course. San Francisco: W.H. Freeman. ISBN 978-0-7167-2866-5.
8. Hannig, G.; Makrides, S. (1998). ‘Strategies for optimizing heterologous protein expression in Escherichia coli’. Trends in Biotechnology. 16 (2): 54–60. doi:10.1016/S0167-7799(97)01155-4. PMID 9487731.

What is gene cloning and how does it work?

DNA cloning is the process of creating multiple identical copies of a piece of DNA extracted from an organism. The very first step of making insulin is the synthetic creation of this human insulin gene. The human insulin gene is at the top of chromosome 11 in humans. Firstly there is a double-stranded DNA, if a part of this DNA has a gene that we would like to clone, the first thing that has to be done is to cut the gene out, this is done by restriction enzymes, which are found naturally in bacteria. Restriction enzymes attach onto the DNA strand and identifies the genetic sequence and ‘cuts’ it in the right place. Now you will be left with the gene. Now we paste this gene into a plasmid using another enzyme called DNA ligase. This then forms a recombinant plasmid that contains our desired gene that we wanted to clone. A plasmid is a piece of genetic material that sits outside of chromosomes that are capable of replicating identically. Once we have “cut” out our gene we then “paste” it into a plasmid using a process called transformation, this plasmid is now genetically modified. Now we have a plasmid which we then insert it into a bacterial cell. Once the plasmid is “pasted” into the bacteria, the bacteria will now be able to survive and reproduce identical copies of bacteria that also contains the new gene and plasmid this is done by a process called binary fission.

What are the possible uses for genetic cloning?

There are three different types of artificial cloning which are: gene cloning, reproductive cloning and therapeutic cloning. One of the main reasons for cloning genes is to gain information about the nucleotide sequence of a gene. Another main reason to why someone would want to gene clone is to manipulate a gene. Meaning to change the gene’s DNA sequence or to merge different DNA molecules together. Cloning genes can help cure many genetic disorders such as cystic fibrosis and immunodeficiency (SCID).When plasmids are created to carry a normal version of a gene that is nonfunctional in cystic fibrosis, these plasmids can help the functioning of patients lungs who have cystic fibrosis. Plasmids are delivered to the lungs of cystic fibrosis patients, lung function becomes better and improves the ability of airway secretions to kill bacteria. Helping to cure these disorders with the help of gene cloning make huge impacts on people’s lives. Gene cloning for medical purposes helps a lot of people. Cloning can also be used to clone different species which have become extinct. When gene cloning is done it can be useful for many different processes, For e.g, detection of diseases, replacement of defective genes. Over many many years gene cloning has been beneficial for scientists to further investigate the genes that are inside our bodies.

What is type 1 diabetes & Insulin?

Diabetes Mellitus type 1 is a condition in which the pancreas which is located behind the stomach, produces little or no insulin. Insulin is a hormone made by the pancreas. Inside your pancreas there are small clusters of cells called islets of Langerhans more commonly known as islets. People who have type 1 diabetes immune system attacks the insulin producing cells therefore damaging them and stopping the production of insulin, which your body needs.The main purpose of insulin is to lower blood sugar known as glucose for energy. Eating causes blood glucose levels to rise. Cells (known as beta cells) are released from your pancreas and signaled to produce insulin into your body. Insulin allows cells to absorb glucose allowing it to be used as energy. Insulin helps to remove sugar from the blood. Insulin also helps to store sugar in the liver and muscles and releases it when your body needs it for e.g, during exercise. When your body does not produce enough Insulin, cells are not able to absorb glucose and used for energy, as a result your body breaks down your fat and protein stores as an alternative way to provide energy. When your body can not produce enough insulin this causes high glucose levels which can cause long-term complications if the blood sugar levels stay high for too long. For a person who has type 1 diabetes it is important for them to keep their glucose levels at a healthy range. By taking insulin as well as eating a healthy diet and exercising regularly you can keep your blood glucose levels within a healthy range. The exact cause of type 1 diabetes is unknown, but in most people with type 1 diabetes, the body’s immune system which normally fights harmful bacteria and viruses mistakenly destroys the insulin producing cells in the pancreas.

Link between genes, chromosomes, protein, insulin, how do bacteria grow, make protein

A gene is a small section of DNA which codes for a specific protein. Genes contain information that our bodies need to make chemical’s called proteins. So, for e.g one gene will code for the protein insulin,which has an important role in controlling glucose levels in your blood. The INS (Insulin) gene is a protein coding gene. This gene provides us with instructions on how to produce the hormone Insulin, which is needed to control glucose levels in the blood. The human insulin gene is at the top of chromosome 11 in humans. Bacteria are tiny living single-celled organisms. Bacteria is found everywhere, from inside your body to everywhere around you. If bacteria are in the right conditions like moisture, nutrition, pH and temperature then doubling of bacteria can occur quickly. Each bacterium has its own condition that it chooses to grow in. Under the right conditions some kinds of bacteria can double every 20 minutes. Bacteria reproduce very simply and fast, by dividing and splitting into two, two becomes four, and the dividing process continues. DNA codes for proteins, the cell sends enzymes to copy parts of the DNA that codes for an individual protein. That’s then transported out into the cell, where it is paired with a ribosome, and translated into a protein. The first protein drug to be produced commercially in bacteria was insulin. To produce a protein in bacteria, “you first need to clone the gene that encodes it, then introduce the vector containing your gene into bacteria”. It is very important to make sure that the vector you clone it into is an ‘expression vector’. By including this bacterial promoter, you are giving the bacterium orders to make a protein from your gene of interest. You are basically ‘tricking’ the bacteria into creating a foreign protein. In today’s society bacteria and other organisms are used as biological ‘factories’ to make protein medicines in large amounts, this is one of the easiest ways to produce a lot of your desired protein.

Economic advantages and disadvantages:

Animal Insulin, how is it made, how much does it cost?

Animal insulin was the first type of insulin to be administered to humans to control diabetes. Animal insulin is taken from the pancreas of animals, commonly from pigs (porcine or pork insulin) and cows (bovine or beef insulin). The discovery of insulin happened in 1921, by a Canadian surgeon named Frederick G. Banting, his chemistry assistant named Charles Best, and John MacLeod. In 1921, Banting and best removed a pancreas of a dog then they “ligated” the pancreas of another, stopping its nourishment. After removing the pancreas from a dog they then threw the pancreas into a blender with some saline ice. And then reinjected the dog which now has diabetes, with its own blended up pancreas.The procedure worked. The dog did not die straight away, it recovered some of its strength. These two men discovered that injecting the dog a few times a day kept the dog alive and healthy. Betram Collip later on began helping these men, and they also started using bigger pancreas’ found in cows. Collip helped to purify the insulin to be used for testing on humans. Banting and Best were confident of their discovery of Insulin and decided to test their findings on themselves. After their testing had been done, their Insulin was ready to be used on patients. After testing the insulin of different patients it proved to be a success, and Banting and MacLeod were awarded the Nobel Prize in Physiology or Medicine.

The first “sale” of insulin was just for $3 (Canadian), not for a vial of Insulin but for the intellectual property of the drug. For $1 the researchers assigned their patent rights to the University of Toronto. In 1941 it was reported by Mr. Eli Lilly that Insulin cost 7.5 cents per day for diabetics. A bottle of pork insulin in 1972 cost $1.49. In Atlanta in 1974-1976, a couple bottles of insulin (NPH beef and pork) costed $2.50. It cost $20-25 for a month of insulin supplies. One vial might last a patient less than two weeks.

Nowadays the price of insulin has increased significantly. Rapid acting insulin can be anywhere from $93 for a vial of Novolin R, to $275 per vial of Humalog. That is a significant increase from a vial of Insulin for a cost of $1.49. People with diabetes can require 1-6 vials of insulin per month.

6Nowadays the price of insulin has increased significantly. Rapid acting insulin can be anywhere from $93 for a vial of Novolin R, to $275 per vial of Humalog. That is a significant increase from a vial of porcine insulin Insulin for a cost of $1.49 in 1972 . People with diabetes can require 1-6 vials of insulin per month.

Production costs: There are different costs between producing gene cloned insulin and animal insulin, because they are both made differently. Manufacturers have to buy raw materials, pay workers, and buy equipment. A paper produced in 1995 focused on the commercial production of insulin using E.coli bacteria. Researchers found that the total cost in making enough insulin to treat one patient a year costed $33.60. Most of this cost is based around the steps of purification of insulin and the fermentation process. Production prices have increased since this paper was recorded due to the new DNA technology in our society, the development of more and different equipment, and the public’s demand for genetically produced insulin. A 2018 study on the yearly costs for the production of insulin reported that the estimated biosimilar prices were from $48-$71 per patient per year.

When the first kind of human insulin hit the market in 1982 animal insulin production declined. With the creation of gene cloned insulin took away any concerns about transferring any possible animal diseases into the insulin. There are now very few companies which still sell animal insulin one of them being ‘Wockhardt UK’ a company which sold porcine and bovine insulin for many years. In 2017, ‘Wockhardt UK’ decided to discontinue the production of bovine insulin due to “global bovine insulin raw material unavailability, the company has been left with no choice but to discontinue the Hypurin® Bovine insulin range”. This company still sells porcine insulin to this day and many years to come but just not bovine insulin. Due to this company’s unavailability of raw materials to make it, the cost of production to make animal insulin would have increased, due to the rising demand of cow pancreas’. Due to the inaccessible resources, insulin companies would of wanted to change to producing insulin from gene cloning. Compared to gene cloned insulin, animal insulin would of had a higher cost of production due to the demand in resources.

During the days of creating pork insulin, it took more than 2 tons of pig parts to produce 8 ounces of purified insulin. This goes to show the very small amount of animal insulin we get out of such a large amount of animals. Breeding and raising of livestock also have to be considered when looking at the total production costs of making animal insulin. Eli Lilly (past manufacturer of animal insulin) needed 56 million animals per year to meet the USA’s demand for the drug. With the help of microorganisms we use to create gene cloned insulin today provides more of the hormone. Bacteria multiplies at rapid speed, so for producers it is easier to make insulin from gene cloning than animal insulin. Without the discovery of gene cloning to produce insulin, animal insulin resources would definitely not have met the public’s demand for insulin.

Everybody’s preference and needs for which Insulin they use varies. One of the most crucial parts in picking which insulin to use for many people would be price which would be a massive factor for some. For one vial of insulin can cost from anywhere between $25 to $417, (depending on if you buy standard, short acting or rapid acting insulin). For many people around the world people can’t afford insulin. Many families whose children suffer from diabetes find it hard to find the funds for insulin. Some families have even had to create GoFundMe pages to fundraise for the medicines. There are even some reports where patients were rationing insulin, and in some cases even dying because they couldn’t afford the drugs they needed to survive. This just goes to show how expensive these medicines really are. People in developing countries are deprived of these medicines. It is proven that gene cloned insulin can be double the amount of animal insulin or even triple for some kinds of gene cloned insulin. For a standard vial of porcine insulin it can cost anywhere from $60 and $90. Even though this is slightly cheaper than gene cloned insulin, there are still many additional charges that come with animal insulin.

In 1998 the manufacturing of beef insulin for human use was discontinued in the USA. In 2006 the manufacturing of porcine insulin for human use was discontinued in the USA. USA has 9.4% of the world’s population who have diabetes, many of these people need to buy insulin. Animal insulin is a lot harder to attain. For people in places like the USA they have to import their animal insulin, and it then has to get checked by the FDA and they have to decide if they will let it into the country.This would result in extra shipping and handling fees, and there is no guarantee that a mistake could happen during the journey. Gene cloned insulin is much easier to get your hands on and a lot more popular, according to the Eli Lilly corporation, “in 2001, 95% of insulin users in most parts of the world take some form of human insulin”, this shows that human insulin would be a more popular option for many people. Gene cloned insulin is obtainable through a prescription from your doctor. Gene cloned insulin can even be brought from a supermarket called Walmart. For some patients, it would be easier to buy gene cloned insulin over animal insulin even though gene cloned insulin is proved to be more expensive. Synthetically made insulin is proven to be easier because the colonies of bacteria are produced at such a rapid speed, meaning there is less resources needed to make insulin and more of the desired insulin, this way in which compared with insulin made from pig and cow pancreas’ need much more time, effort and resources involved with the process, and the result is a small amount of insulin for how many resources would be used.

Others P.O.V’s of using gene cloning to produce insulin

Everyone has different thoughts when it comes to the idea of using gene cloning to produce insulin. Some people think that using gene cloning to produce insulin goes against the laws of nature, or against people’s religious beliefs. Many others are mostly concerned about safety issues. Using gene cloning can also be beneficial to people is so many ways, usually people in the past were only expected to live 4 more years after being diagnosed with diabetes, but now that we have the new DNA technology to produce human insulin, humans can be expected to live a normal life, with a normal life expectancy. Many people see it as a great and effective way to improve people’s lives.

PROS:

12Sverker Johansson is a Swedish Physicist who sees genetic engineering as a sign of progress and an opportunity to make the world and the lives of humans and animals better. He also believes that genetic engineering can make pharmaceutical products more effective. “There are already improved versions of insulin for diabetics and human growth hormones on the market thanks to genetic engineering”. Sverker finds all the positives of genetic engineering, from making fruits even bigger and better than the ones before to improving the lives of so many individuals because of genetic engineering to produce insulin, ‘I believe that genetic engineering, can benefit people in several ways, for example, scientists made artificial human insulin with the help of recombinant DNA technology”.

CONS:

The Catholic Church’s views on genetic engineering: There are many different responses to genetic engineering in the catholic faith:

One of the reasons would be ‘anti-god’. Many Christians believe that God created all living beings on earth (Colossians 1:16), and by genetically modifying creatures we are going against the authority of God. “The belief that God should have ultimate power and we should not be altering nature is what some Christians believe should have us halt the progression of human GE”. Most of the people in the Catholic Church agree with gene therapy to treat diseases but not therapeutic cloning. They believe that “without certain diseases, it would be possible for more people to live longer, healthier lives”. Gene therapy is used to cure many different diseases, the Catholic Church agrees with the use of gene therapy but not so much with therapeutic cloning (human cloning).

(Statement from Pope John Paul II) “Reproductive and therapeutic cloning seems specious. Both forms of cloning involve disrespect for the dignity of the human being. In fact, from an ethical and anthropological standpoint, so-called therapeutic cloning, creating human embryos with the intention of destroying them, even if undertaken with the goal of possibly helping sick patients in the future, seems very clearly incompatible with respect for the dignity of the human being, making one human life nothing more than the instrument of another”. The Pope believes that God is the one who hand crafted our souls from the moment of fertilisation, therapeutic cloning goes entirely against this. Christians think that the dignity of the person is being overlooked.

Gene cloning is a biotechnology in which a section of DNA is isolated and extracted before being cloned using bacterial plasmids. To execute this process, a restriction enzyme isolates a specific gene from a strand of DNA and the plasmid, and then cuts the gene creating complementary sticky or blunt ends. These are joined to form recombinant DNA, which is then inserted into bacteria via heat shock to be cloned. Gene cloning is used for many reasons such as to create therapeutic proteins and to create DNA libraries. It can can create therapeutic proteins such as insulin in higher quantities than previously by inserting the recombinant DNA into E.coli. DNA libraries are created which can help to work out long DNA sequences to create maps of chromosomes.

It is also thought in the future that scientists may be able to utilise gene cloning to create tissues and eventually organs out of a single cell. Implications and Issues with Gene Cloning As with all biotechnologies there is a great amount of social implications and ethical issues with gene cloning. This technology greatly benefits medical society as it allows for; greater amounts of life saving proteins to be synthesised, increased availability of replacement organs, and the process of gene therapy, where normal genes are inserted in place of defective ones. However, socially it could have damaging effects, with the ability to create designer babies, as attempted by He Jiankui when trying to modify babies to disable the CCR5 gene, and other ethical implications such as the experimentation on animals. Furthermore, there is a great many ethical issues such as the potential for cell degradation from creating too many clones, and the bacteria used in cloning may have antibiotic resistance which could be transferred to other bacteria via horizontal gene transfer.

How does Gene Cloning affect Earth’s Biodiversity? Gene cloning has the potential to greatly deplete Earth’s biodiversity in the long term, as it contributes to the dominance of particular alleles in a species. It can be implemented to replace copies of genes leading to the disappearance of defective or missing genes, so ‘fixing’ DNA sequences, as in the experiment by He Jiankui, can cause loss of biodiversity. Additionally, the increased availability of insulin has led to increased usage, thereby increasing biodiversity as it has allowed those suffering from Type 1 diabetes to live long enough to reproduce thus passing on genes that increase risk of diabetes, like some variants of the HLA-DQA1, HLA-DQB1, and HLA-DRB1, to their offspring.

Furthermore, in the process of cloning genes, often bacterial plasmids are given antibiotic resistance to grow a culture of the pure gene effectively. This could escape into the general bacterial population as bacteria can pass plasmids to others through horizontal gene transfer which speeds up the spread of this trait, making it a dominant trait within that population. This could result in antibiotics becoming ineffective, leading to doctors being unable to effectively treat bacterial diseases which could eventually decrease biodiversity due to possible extinctions. Therefore, gene cloning increases and decreases biodiversity, however in the long term it may lead to great loss of biodiversity. Does Gene Cloning have the potential to change populations forever? Gene cloning very much has the potential to change populations forever. Although it has many benefits it also has disadvantages such as the ability to greatly deplete the Earth’s biodiversity. This loss of biodiversity would greatly change populations forever as it could lead to the extinction of certain populations as as previously described.

It could also positively change populations with endeavours like creating organs from single cells which would allow organ transplants to be readily available, thus increasing the number of transplants given, resulting in an increased life expectancy within populations. However there are more negative possibilities like the prospect for designer babies, which has has become a concern in the scientific community since the work of He Jiankui was released. His experimentation shows the possibilities of gene cloning, via gene editing, which could change populations forever in a deliberate way.

Lastly, synthesis of therapeutic proteins, although a positive development, could have the possibility to negatively change populations, the increased use of insulin means that a larger percentage of the population develops Type 1 diabetes. This could be due to the certain variants of the aforementioned genes being dominant alleles, which could lead to much of the human population developing diabetes, thus changing populations forever. Ultimately, gene cloning has great potential to change populations forever.

Humans have been breeding dogs for at least an estimated 14,000 years, it is said to believe that the evolution of the domestic dog evolved from the wild grey wolf (Canis Lupus.) As humans began domesticating dogs, they had begun to favour specific traits such as dogs with particular physiques and temperaments, hunting skills, intelligence, behavioural traits and even the simple companionship between man and dog.By selecting the most favourable dogs to breed, humans were unconsciously using artificial selection to advance the breed of dog.(Canis Familiaris.) Over time humans have since begun manipulating dogs for that purpose, for the purpose to advance the characteristics of interest in the hope that their offspring inherit and display those desirable characteristics,therefore made to meet the need for human demand.Through the use of artificial selection they were able to create a vast variety of dog breeds. Resulting in many organisms/individuals to vastly differ in appearance, inhibiting particular genetics/DNA.Therefore to embed different qualities essential to excel for their purpose.This potentially causes variation to a species or gene pool which is essential for the survival of an organism.Since then biologists have also recently begun genetically manipulating dogs for desirable traits such as increased muscle mass, stronger running ability, also to create dogs with other DNA mutations, including ones that mimic human diseases for biomedical research purposes. Genetically modifying dog breeds and the use of genetic engineering technology however, has had wider biological implications.

Selective breeding, also referred to as artificial selection is a process used by humans to select two organisms with particular genotypic and phenotypic traits in order to produce favourable offspring who will inherit and display those desirable traits or genes, it is these genes that will then be passed on throughout future generations.In this case humans had selected the two favoured breed of dogs one being female and the other male.They had then crossed them together, therefore resulting in a new generation of the favoured offspring, making it a successful reproduction. This will then in turn advance the organisms of the future generation. For instance each of the favoured offspring has resulted from a female egg fertilised by male sperm, this means that only the best or most favourable offspring will reproduce and the gene pool of the next generation will have a higher frequency of the favoured genes.Over time the traits of the favoured offspring will be displayed more frequently throughout future generations as the favoured offspring reproduce continuously, therefore being passed down throughout generations, continuing in a repetitive cycle.

Through the use of selective breeding it can also be used to eliminate undesirable traits from a breed which can come as an advantage in some cases but can also have negative effects. When one specific trait is desired, certain breed of dogs are often used (manipulated) to create the needed offspring. If that gene pool of dogs results in only that offspring produced with only those desired traits then each subsequent generation will lack other qualities because there is a decrease/lack of genetic variation present. When a gene pool of organisms all have identical desired traits due to human demand eventually outside genetics must be introduced into the gene pool to preserve the traits If that were to occur, however by doing so this could result in the outside genetics to also then dominate the desired traits and reduce the current gene pools appearance/qualities. Therefore result in the removal/deletion of desired traits/genes from the gene pool causing for what is known as genetic drift.The chances of this happening would be highly likely for a gene pool of inbred organisms.

Selective breeding has become easier over time as people as well as breeders have found a more effective way to figure out an organism’s genotype, this was first carried out using a test cross, By crossing one organism that is expressing the dominant form of a trait with another of the same species that is homozygous recessive for the same trait, it is then possible to determine the offspring’s genotype from the traits expressed in those two species. By doing so it is also then possible to determine or have an ideal concept of the genotype of other organisms from the traits expressed in the offspring.Therefore by crossing one organism of the same species that both possess either homozygous dominant or homozygous recessive genes, breeders can now more effectively cross two organisms of the same species and can then almost guarantee a purebred offspring.

However the use of selective breeding and offspring inheriting “favourable” genetics cannot always be 100% guaranteed as selecting from a breed of dog that only has one or a few desirable traits is not always reliable, this is due to the hidden mutations and hidden genes that can occur, although the phenotype that may seem to be suitable, the genotype (and therefore phenotype) of its offspring may not be suitable. If a female breed of dog was to have a recessive gene in its genotype that was not expressed in its phenotype, and her eggs were fertilised with the males sperm, and the male were to also have a recessive gene in its genotype then it would be highly likely that there would be a chance of the offspring being homozygous recessive, however this would also mean that the particular phenotype expressed in the female breed of dog would also not be expressed in the offspring/new generation.This would have a negative effect on the breeder as they wouldn’t realise the result, therefore the offspring being homozygous recessive without the breeders knowledge.

Methods like Marker Assisted Selection (MAS), otherwise known as marker aided selection is an effective method that is now used for indirect selection of a genetic determinant of a trait of interest. (MAS) can be useful for traits that also exhibit low heritability, traits that are difficult to measure, or are expressed in late development. The process of Marker Assisted Selection first include mapping the genetic trait of interest or quantitative trait locus (QTL) of interest by developing various techniques and then use this information for marker assisted selection. If both the marker and gene are located far apart then the possibility of selected individuals being recombinants will be highly likely. The markers used should be closely linked to the gene of interest to ensure that only a minor fraction of the selected individuals will be recombinants. Generally not only a single marker but two markers, preferably RFLP markers are used in order to reduce the chances of an error due to homologous recombination.

Other more efficient methods have been carried out, For instance a source known as Artificial Insemination (AI) is also another effective method that is now used for indirect selection. Artificial insemination is the process of male sperm being artificially inserted into the cervix/vagina of a female dog.The insertion of (AI) is applied to avoid the risky trial and error process of selective breeding.This method is used for the purpose of the potential benefits it provides, these benefits include increased safety of the organism and increased production efficiency. Most males usually produce enough sperm in a single ejaculate to be diluted and extended enough to create over one hundred doses producing many semen samples for reproduction. By using artificial insemination humans are able to more effectively determine the genetic trait of interest providing a more guaranteed result. AI is used for preventing or Eliminating the risk of possible diseases such as brucellosis or CTVT, it can also be useful for traits that display low heritability or lack the heritability of the desired gene.

Since humans have begun selectively breeding dogs, problems are arising from a genetic standpoint. This is because certain breed of dogs are often being selected for specific traits and those traits only. For example once those specific genes in dogs are selected for and are consistently being bred due to human demand, other genetics in a dog species of the gene pool will eventually over time be non existent/removed from the population as the favoured genes become more dominant over the other genes in the gene pool, thus replacing those undesirable genes.Once that gene is breeded out of evolution it is very difficult almost impossible for you to bring that gene back, therefore the gene for that organism that once existed in the previous generation will no longer exist having a major impact on the ecosystem.

Another biological implication of selective breeding is that it reduces genetic diversity. The lack of diversity in a gene pool between each dog and their alleles due to being closely related would increase the chances of organisms to become susceptible to the same diseases/pathogens. An example of this is inbreeding, inbreeding increases homozygosity. This is because inbreeding can result in both harmful and undesirable genes inherited in both parents to become expressed in their offspring having many negative effects. Continued inbreeding would result in an accumulation of recessive genes which can cause inbreeding depression, this is not a state of mind but rather a physical state resulting in low fertility, poor general health such as weakened immune systems and most importantly inbreeding results in negative/undesirable genes increasing the chances of being inevitably prone to disease. This is of major concern when attempting to protect small populations from extinction.The consequence is that little new genetic information is added to the gene pool, thus recessive deleterious alleles become more increased and evident in the population, eventually resulting in the species of the population to be removed from the gene pool.

Other than the use of selective breeding, humans have also discovered another technique that is now used for the manipulation of dog breeds. This process is referred to as Mammal cloning.With the use of genetic cloning technology biologists are now able to more effectively have more control throughout the process therefore having a much more higher success rate allowing them to introduce certain genes into a dog without having to go through the relatively long and unguaranteed process of selective breeding. By using cloning technology it is now much easier for biologists to add/insert certain genes into a species one may not naturally possess. The purpose of this technology is so that scientists can produce genetically identical copies of a gene from one desired organism to another creating the perfect replica so that those specific genes can still be carried on. They had used a method known as (SCNT) referred to as Somatic Cell Nuclear Transfer. Using this technique scientists select the nucleus of a somatic body cell from the desired breed of dog/nuclear donor and then transfer the cytoplasm of an enucleated egg and insert it into an unfertilised egg cell. Once inside the egg the somatic nucleus is reprogrammed by egg cytoplasm factors to become a zygote nucleus in which the embryo develops into a fetus.The eggs are then inserted into the uterus of a female dog who will serve as a surrogate.

A recent study conducted by Chinese scientists used this genome editing technology to modify a breed of dog such as the beagle. This specific breed of dog has been selected/modified for certain beneficial traits to assist with new DNA mutations, human diseases such as cardiovascular disease, Parkinson’s and muscular dystrophy. The beagle came about to explore an approach to new disease dog models for biomedical research. The technique used for the beagle is a form of transgenesis which is the process of introducing foreign deoxyribonucleic acid (DNA) into a host organisms genome. This is usually done through experimental manipulation of gametes/ early embryos.They had created a beagle with double the amount of muscle mass by deleting a gene called myostatin, it had then displayed obvious muscular phenotype.

The scientists had developed/modified the beagles by using a supposedly more effective and accurate gene editing tool CRISPR-Cas 9. CRISPR -cas9 consists of two key molecules that introduce a Change (mutation) into the DNA. This is the enzyme cas9 which acts as a pair of molecular scissors that then cuts the two strands of DNA of the beagle at a specific location in the genome so that pieces of DNA can then be added, therefore the hereditary is now existing as the favourable transgenic gene therefore giving it the ability to express the gene encoded by that gene, it also can be used to remove/knockout specific genes.CRISPR -cas9 consists of a piece of RNA called guide RNA (gRNA). The Guide RNA is designed to find and attach to a specific sequence in the DNA. It has RNA bases that are complementary to those of the target DNA sequence in the genome, this means that the guide RNA will only attach to the target sequence and no other regions of the genome. The cas9 then follows the guide RNA to the same location in the DNA sequence. And makes a cut through both strands of DNA. The cut is then repaired introducing mutation. However not all 20 bases need to match for the guide RNA to be able to attach. The problem with this is that a sequence with, for example, 19 of the 20 complementary bases may exist somewhere completely different in the genome. This means there is potential for the guide RNA to attach there instead of at the intended target sequence. The Cas9 enzyme will then cut at the wrong site and end up introducing a mutation in the wrong location, while this mutation may not have an effect on the beagle it could affect a crucial gene or another important part of the genome.

A biological implication of the beagles that have undergone the process of mammal cloning is that there will be very little genetic diversity within the population as the dogs have been cloned and reproduced all from one same dog, meaning all their genes will be identical. In the same way that if affects selectively bred dogs for example inbred organisms, it will also affect genetically modified dogs in the process. Due to the dogs all having similar DNA they will all be susceptible to the Same things or changes in the environment such as particular diseases, health issues for the individuals, for example most dogs that are selected for, a vast majority of those dogs suffer major side effects such as breathing problems in the pug, problems with their immune systems etc, they end up suffering just to assist with a humans satisfaction, those that are cloned are often deliberately created with genetic defects.The use of genetic modification in the long run could significantly decrease resistance to disease and result in undesired traits rather than improve it like intended.

Another biological implication of mammal cloning is the harmful mutations involved with genetic modification. Somatic cells play a significant role in cloning, when a harmful mutation tends to alter these somatic cells in the cloned organism due to gene modification it could then cause the breed of dog to have lethal consequences such as cancer or genetic disorders. Because of these mutations, the affected cells could then possibly divide without limitation therefore resulting in cancer also allowing the cancerous genes to become hereditary. Therefore increasing the chances of generations to inherit cancerous genes/genetic defects.

All breed of dogs that we see today are a product of selective breeding.There are advantages and disadvantages involved with both selective breeding and mammal cloning.In selective breeding once humans began to manipulate a certain breed of dog,it had then resulted in the dogs genes to become non existent/removed from the population.Once this happened there was no going back, the original breed of dog no longer existed.Once the original/previous genes are removed from the population due to human manipulation those genes would be completely lost, therefore making it impossible to undo that. In mammal cloning it has the possibility of a species to also become scarce, as all cloned organisms would inherit identical genetics, therefore the organisms become susceptible to the same things such as diseases etc, resulting in the species to also be wiped out. Although there is a smaller risk of the cloned species being entirely wiped out as the original species will still exist, even after some of its DNA has been manipulated. However both manipulations still decrease genetic diversity and put the species being manipulated at risk. With genetic manipulation becoming more complex, the use of both selective breeding and mammal cloning will still have its limitations and implications.

“I saw a new world coming rapidly. More scientific, efficient, yes. More cures for the old sicknesses. Very good. But a harsh, cruel, world. ” (Never Let Me Go)

Science. Genetics. Cloning. Human cloning. Human cloning will alter our world forever and will transform it for the worse.

Science has developed throughout the years and humanity has changed drastically from new discoveries. Things like the invention of the telephone, the discovery and medical applications of penicillin, and the creation of the car have shaped the world and impacted our daily lives. Nowadays, scientific discoveries have taken a turn for the worse.

Nobody has managed to successfully artificially clone a human before, but there have been other animals that have been cloned. In 1996 at the Roslin Institute in Scotland, there was a sheep named Dolly, who was the first mammal to be cloned using an adult somatic cell. Out of 277 attempts, only one embryo was able to be implanted in a surrogate mother(the other ones did not survive). This was one of many stepping stones that helped to advance cloning.

Then, only a few years later in 1998, scientists tried to use similar techniques that were used for Dolly for other mammals. Sure enough, they cloned mice, cows and goats. In 2001, they were able to clone an endangered animal using somatic cell nuclear transfer. Using this technique, they cloned an extinct animal, a mountain goat named the burcado, which is absolutely incredible. Unfortunately, the baby died a few minutes after birth due to a lung defect. This was the first successful “de-extinction.” I completely agree with this type of cloning, since it is helping to increase biodiversity. Recently, Mu-ming Poo along with a team of Chinese scientists have cloned macaque monkeys named Hua Hua and Zhong Zhong. Poo said that the combination of two types of technology, SCNT and gene editing, would create ‘ideal nonhuman primate models” for studying the origins of different diseases, and possibly drug screening.

The monkeys’ successful cloning began to raise the question: “Could we clone humans?”

Many people have been naively asking whether it is possible to artificially clone humans. Well, the obvious response to that is that we can. I think that these people are missing the real question, of whether we should clone humans. “Cloning people, what could be so wrong with that?” you might ask. And so the argument begins. To clone or not to clone, that is the question.

When I was a little girl, my beloved grandfather almost died from lung cancer. He was a jolly man, a lively man. When he got sick, I remember visiting him and seeing that the man that I once knew was gone. Instead, a ghost had taken his place. This ghost did not feel like playing catch or telling me jokes. This ghost sat in his bleak hospital bed in his bleak room all day, staring at the wall emptily and somberly. I was devastated, desperate to get my grandfather back and get rid of that ghost. I was thinking, what if someone took his place? I sat down on the hospital chair to his right and thought: What if someone, like a clone, could give him a new set of lungs, and he could become himself again? With a new set of lungs, he would be that happy man again, my funny and sweet grandfather. A few seconds later, the doctors walked through the door and asked me if I wanted a clone to give him a new set of lungs, which would save him. Immediately, I said yes. And my grandfather is still living and laughing, at 93 years of age.

Now I realize the error in my thinking and in my decision. Although I agree with the fact that cloning could save many people, most people are still forgetting or do not understand the numerous consequences if human clones would be created. If cloning were to become something that is available to the public and clones would be a part of our daily lives, this would be a disaster of inexplicable proportions. Although it may be hard and it may be frustrating, killing clones to use their organs is unacceptable, no matter how tempting. Creating clones in the first place is a bad idea. You can look at it from any point of view, whether it is through the environment or politics, and can immediately see that none of these things end well.

In terms of the environment, and cloning would end up hurting the environment more than it would help. We already have a planet that contains about 7.5 billion people, and there are hundreds upon thousands of people that are living in very poor conditions and in overcrowded areas in poverty, malnourishment, and with less than two dollars a day. Do we really need more people on the planet? Do we really need more clones to drive cars and contribute to global warming? Even though Donald Trump says it does not exist, that is not true! Climate change would get even worse with clones. We do not need more people contributing to our already large ecological footprint.

Furthermore, there are many political ways that clones would harm society and the world as a whole. There might be negative feelings from the press and from governments towards clones and cloning as a whole, and this would create a large source of controversy. This would create a stigma around clones, and a divide between citizens, which would create more issues than we need right now.

Not to mention the overall harm that cloning would have on society. Logically, the government would invest money into cloning, since in the future, they would be able to use clones as slaves and workers, so they would not have to pay them and this would benefit them in the end. These ignorant and avaricious people are only looking out for their own well-being while making us suffer like fools. By putting all of this money into cloning, they are ignoring real issues, such as poverty, food and water security, climate change, and inequality. These two-faced liars only pretend to care about issues so they can get elected, but that is where the sympathy and promises end. These so-called “leaders” hide out in their fancy offices, sheltering themselves like cowards from the issues in the world and scheming on how to make more money than they already do. Do they really care about the well-being of the clones as long as they get richer? No. Do they really care that these clones are being treated unfairly? No. Do they really care that developing cloning will neglect other pressing issues? No.

Overall, having clones would create more harm than good for both humans and clones.

Finally, if this were to happen and if there were going to be human clones, this would eventually lead to us trying to take power over them and us trying to control them and use them as resources because that is what humans do. We try to control other people, it is just what we do. “Why does it matter that we try to use them?” you ask. Well, let me explain.

Many people are saying that we should clone people so that we can use their organs to survive, but this is wrong. This is evil, this is doing everything that we are standing against. We stand for animal and human rights, but we want to hurt other living beings. We take such good care of our technology and treat it with respect, but would kill our own creations. Either way, humanity is being hypocritical by killing the clones.

So, again, should we clone, or not? My answer: No cloning. Just no. If we clone humans, this will lead to people taking the clones’ organs and many other consequences. Now, why is it so bad to clone people to take their organs?

If you think about it, what really is a clone? They are people with the exact same DNA as us. However, they are created artificially. This does not make them any less human. Clones think like us, hate like us, love like us, and feel like us. So, they should have the same inalienable rights as us.

It is the same thing as saying that twins, natural clones, should have fewer rights than other babies. People would be outraged! This would not be accepted in society! So, this should not be the case for clones.

Since it is clear that humans possess identical qualities to clones, they should also have identical rights and identical treatment. No one in their right mind would go around killing humans for their organs without their consent. Clones should be no different.

And anyway, who gives us the ultimate power to kill other beings, no matter who or what they are? No one. This may be a surprise to some people, but human beings are not superior to other living organisms(Wow, right?). The one and only thing that makes us equal is the fact that, in the end, we all die, even if this sounds very morbid. Rich or poor, animal or human, all living things will all die at one point in time, and for that reason, we are all equal.

Because we only have limited time on Earth, no one should have the right to kill other living beings. Even if humans do kill other humans, it does not mean that that is the right thing to do. These people either work for the military and are oblivious to what is right and wrong, or are out of their minds. Humans do not have the right or the authority to kill other living things, other than for predation(ie. survival), and this is clearly not the case. This is the abuse of power, this is taking advantage of other beings, and this is being worse than any other animal.

Even if people were considered superior, because of “language” and “effective communication” and whatnot, that still does not give us the right to kill other living organisms. Listen to our good old friend Pooh: “‘If people were superior to animals, they’d take good care of them.’” I know, he’s a fictional character, but he is pretty smart. If we think that we are better than other beings, we would take the responsibility to help to conserve their lives, not take them away. By killing the clones and taking their organs, we are just proving that we are at the same level, if not lower, than other beings. By killing an innocent creature who has never done anything to anyone and cannot defend itself, we are proving that we are sadistic and evil creatures.

Finally, we should not clone people just to harvest their organs because this would create a ripple effect all throughout society. Up until now, people have taken some measures in order to keep their life, because it is part of our DNA to not want to die, although there have been a few numbskulls that have come pretty close to death from adventure or taking risks. Without this part of our DNA, how do you think cavepeople would have survived numerous predators? With all the idiots on our planet, our species would probably be extinct by now if we did not have this instinct.

However, with the addition of the cloning program, all of this might change. People might start to take their lives for granted, even more than they do already, now that they have a new way to stay alive. It is not as though they would have a problem getting the organs, and they would not feel bad for killing the clones, because they wouldn’t know or wouldn’t care.

It is like when you go shopping at the mall. You walk into stores and shop at stores like Forever 21 and Zara, even though you know that they use sweatshop labor. Even though you know there are people suffering in sweatshops all over the world to make those clothes, you buy them anyway. Think about it. Everyone does it.

However, doing this does not make you a bad person. If you live in a country like Canada where you are not exposed to these issues and this is not the norm, it is very difficult to care about them. You cannot have feelings for something that you do not know all the facts about, and you cannot really care about anything if it is not in your own interest. By not caring about the clones, people are following what is in their own interest, which is getting the organs and living longer. Selfish? Definitely. Improbable? Absolutely not.

So, not only would people not care about what happens to the clones, they would not care about what happens to them, since they would have something to fall back upon. People would have no sense of urgency, and no sense of caution. This would have disastrous effects on our society.

When I was younger, my sister and I loved to jump on the bed. A lot. So, although we knew that what we were doing was dangerous and we might get hurt, we decided that we would put a couch cushion on the ground, just to be safe. Sometimes we fell on the cushion, sometimes we didn’t. But the point of this story is not to encourage you to use clones as cushions on the ground when you are jumping for donations.

By having the cushions, or the organ donors in this case, we would do some stupid things, even if they were not 100% safe because we knew we would have something to fall back on. Now, imagine everyone was doing stupid things and putting their safety at risk because they had something to fall back on. Society would collapse in on itself, and everything would turn into chaos.

Here’s the big question: should we clone, or not? The only reasonable answer is that we should not clone. Why would we create sentient beings just to kill them? This is just inhumane and quite frankly, insane. Why create people and make them suffer?

These scientists might have book smarts, but they fail to see how their discoveries will really impact the world. Getting degrees in biology and anatomy won’t really help them see what is inside people’s hearts, which is life and passion. Clone or original, animal or human, we all have life inside of us. Would you rather survive or live? Would you rather spend your days chasing death than barely making it out alive? Or would you rather spend your days living your life to the fullest, using whatever time you have on Earth wisely?

Science has to move in the right direction, the one where we prevent suffering, not cause it. Although it may not seem like it and although it is a cliché, every individual has a say in how our world will adapt with the development of cloning, since it is inevitable. If you stand up for what you believe in and tell people how cloning is wrong, you can make a change. Spread the word, and let others know what you think! In the end, life is a journey; it’s your choice whether you want to cowardly hide out in the shadows where there is no light, or take center stage and fight for what is right.

Science. Genetics. Cloning. Human cloning. Human cloning will alter our world forever and may transform it for the worse. That is unless you decide on an important role for the clone.

Even if I said yes to cloning, you don’t have to. But I will.

Nowadays, Clone is a word that a new commonly used in many contexts all around the world, especially in the United States. Scientists have discovered a way to bring back extinct animals. Many people dream to extend life for their loved ones and being able to express the hidden feelings in their minds after their loved one has disappeared from their life. The idea is that humans might someday be cloned in the future, but there is some scientific concern about cloning that may change the world forever. Even though scientists have discovered a way to bring back human life by Cloning but it’s not 100% sure that the scientist will lead to successful cloning in the present and there are many disadvantages to cloning as well.

Many people have heard about cloning since Dolly the Sheep has shown up on the screen in 1996. The process of scientists that worked hard from 1996 – 2003, Dolly the sheep has become the first successfully cloned mammal in early 1996, at the Roslin Institute in Scotland. Dolly the sheep was cloned by Keith Campbell, a British cell biologist who helped usher into one of the most famous animals in creation (Margalit, 2012). Keith Campbell was born on May 23, 1954, in Birmingham, Unified Kingdom. He is a professor of animal development at the University of Nottingham who was a member of the first cloned mammal team in 1996. In November 1999, Keith Campbell has become a professor of Animal development at the University of Nottingham. At the age of 58 professors, Keith Campbell commits suicide by tying a belt around his neck and hanging himself from a ceiling beam in his bedroom. He died at the age of 58 on October 5, 2012. The process of Dolly the sheep contains a lot of process and time to succeed in this project. Dolly was created by the cells taken from the udder of a Finn Dorset ewe and placed in a culture with a very low concentration of nutrients, in this step the cells will stop dividing and switch off their active genes. Secondly, the egg cell is taken from a Scottish blackface ewe. The nucleus is sucked out, leaving an empty egg cell containing all the cellular machinery necessary to produce an embryo. Two cells are placed next to each other and an electric pulse causes them to fuse together like soap bubbles. After about six days, the resulting embryo is implanted in the uterus of another blackface ewe. After all the previous steps, the pregnant blackface ewe will give birth to a baby Finn Dorset lamb named Dolly (Jee, 1997). Presently, There are some questions from people around the world “Why do scientists clone?”. Nowadays, the main reason for cloning might include replacing lost or deceased family pets and repopulation of endangered or extinct species. Currently, the new cloning technologies have sparked many ethical debates among scientists, politicians, and the general public (Craig, 2019). The first study of cloning took place in 1885 when a German scientist Hans Adolf Eduard Driesch was able to create a set of twin salamanders by dividing an embryo according to the genetic science learning center. Since the experiment of Hans Driesch has reached success there have been many breakthroughs in cloning since then. Cloning can be useful for clinical trials and medical research in the future.

“Clone” is an exact genetic copy of an organism, that may be naturally occurring or created in the lab. Some people might have thought typically think of organism cloning, but actually, there are three types of cloning processes that were used in a lab which are Molecular cloning, Organism cloning, and therapeutic cloning. Firstly, Molecular cloning or also called gene cloning will focus on making identical copies of DNA molecules in the chromosomes. Secondly, Organism cloning or also called reproductive cloning will involve making an identical copy of an entire organism such as bacteria and some other plants create offspring that are genetic to the parent. Lastly, Therapeutic Cloning involves the production of stem cells by involving the cloning of human embryos, but in this process sometimes those embryos are eventually destroyed in this process. It’s true that cloning is a new scientific technology that develops our resources, but there are risks to cloning as well. There are some groups of people around the world that are interested in Cloning but some groups of people are concerned that cloning will upset the nature and the course of evolution. After the process of cloning Dolly the sheep by scientists in Scotland in 1996, several other mammals have been cloned, including dogs, cats, and pigs. Most people have a limited understanding as to what cloning actually is, some think that cloning was about creating a genetic copy of the animal species, but cloning is even more complicated. There are many Negative impacts of cloning that cause an animal to suffer loss or extinction of species, such as Dolly the sheep had a progressive lung disease and severe arthritis. Some people think that if there is a scientific error in the experiment of a scientist it may cause a huge problem for the entire planet, it may cause mutation among humans and organisms that can change the world.

It basically involves a method for developing a replica of tissue, organ or cell which are inherently similar to each other.it occur in nature- for instance, when some cell replicated itself asexually without chromosomal mutation and linkage. In prokaryotes, bacteria can produce a genetically alike copy of itself with help of binary fission. On other hand, in eukaryotes like in humans all cells such as skin cells, digestive track lining cells go through mitosis thus produce clones, except from the gamete cells that undergo meiosis and genetic recombination.

Types of cloning

There are two main types of cloning

Natural cloning:

This type of cloning take place through sexual reproduction and do not involve any kind of genetic makeup alteration. For instance, many types of bacteria and human also undergo this process for reproduction. In bacteria clones are produce through binary fission and their genome split into two genomes while in human identical twins is best example in which fertilized egg split and more than one offspring produce. These identical twins have same genetic makeup but different phenotypes from parents.

Artificial cloning

Replicating genetic material to produce cells or organs having duplicate genetic information term as artificial cloning. The main purpose of this process is to produce the cell, organs or tissues that have desire traits for the research and scientific study purposes.

Somatic cell nuclear transferase

It is a prevailing method. In biomedical research, this is term as replication of any biological substance such as a piece of DNA or a single cell can be duplicated by using PCR. In July 1996, fist artificially cloned mammal DOLLY sheep is cloned with help of novel method named as somatic cell nuclear transferase (SCNT). In this process, firstly genomic information is detached from egg of female then somatic cells are isolated from desire organism which has to be replicated and nucleus cell is injected into host oocyte with micropipette. Then egg, so “fertilized” is stirred to initiate embryonic growth.

Cloning process of DOLLY sheep

There are various types of SCNT cloning such as the:

• Genetic cloning in which copies of gene or DNA fragments is produced.
• Reproductive cloning in which whole animal is replicate. In 1900, German embryologist Hans Spemann firstly reproductive clone a salamander embryo.
• Therapeutic cloning which involve duplication of embryonic stem cell. With help of these cells researchers intend to develop healthy tissue to interchange injured tissues or unhealthy tissue in body.

In biotechnology, this process need a gene of interest, a vector which carry desire gene, host which give suitable environment to desire gene to duplicate, and medium for development of host strain. However, one method which is under spot light due to its ethical debate is generation of replicated embryo specially of those which are human based and genetically duplicate to those from which they are originated, and frequently use of embryo in research, reproductive or in therapeutic methods.

Scientists around globe endure to debate over benefits of this in anticipations of being able to research and study it more comprehensively. According to an estimate more than 30 countries allotted a ban on it that involve human generative replication. But out of them some countries such as China, Sweden, England allows this for some beneficial reasons that does not depend upon human based reproductive replication.

Benefits

The major benefits involve

Organ replacement

In this scientists take minute quantity of desire organ part for development complete new functioning part of tissues and organs for therapeutic purposes. If labs can replicate only those tissues and organ parts which are necessary than it will minimize the ethical and moral problems linked with complete organism cloning. It is also beneficial because there is a number of patients on organ donor waiting list.

For the production of stem cells.

These are used to create, stabilize and respire damage organs and tissues in body. Some researchers are doing work in order to develop stem cells that are similar to recipient. These cells can also helpful in generation of complete organ and for study and understanding disease to create possible treatments.

For growing genetically cloned lab mice for research studies.

A researcher can understand a human disease by experimenting on animal models such as mice. Mostly animal model made engineered by causing disease developing mutations in their genes but this method is very time consuming and errors and trails are needed on number of generation breeders. Cloning can help to overcome this problem by making a transgenic animal model which result in large number of animal models for study.

It may be helpful for bringing back the extinct or angered species.

In theories it may be possible to bring back an extinct or endanger specie. for this scientist need a source of DNA from well preserved extinct animal source and a closely related animal species which act as egg donor and replicated mother.

For cloning a livestock for food.

There is way progress to replicate a livestock for food such as cattle which are main source of milk and meat.

Cloning help sterile couple too have baby.

Infertility problems can also be overcome by using this method. In this process stem cells are isolated from male parent and then it is inserted in egg and subject to develop as an embryo. The resulting offspring have characteristics of both parents.

Disadvantages of cloning

Along with benefits there are some demerits of as well such as

1. It is the copying of identical genes that in result leads to minimize diversity of genes?
2. As human clones are alike they all are potential risk of getting infected with same pathogen this will result in great disaster.
3. Another disadvantage of cloning is in-breeding, as all have same genotype and reproducing among themselves will cause extinction at end.

Therapeutic cloning

Therapeutic cloning involves methods used to isolate stem cell for research study and also for recovery of degenerative disorders in future. Stem cells are cell which through differentiation method generate different specialized cells that may be totipotent to multipotent. stems cells used in therapeutic cloning purpose are embryonic, bone, blood, skin and umbral cord cells. The process involve is enucleation of one cell and inoculation of somatic nucleus in that cell. After this inoculation in vitro reproduction of cell is initiate to isolate stem cells with desire traits of somatic cells that are further used for therapeutic purposes

Cloning possible uses in therapeutics

it can be helpful in deteriorating disorders such as in DIABETES TYPE 1 or in future may be able to cure the degenerative mental disorders such as Alzimher and Parkinson diseases.

Diabetes type 1

It is an autoimmune disorder that is triggered by fabrication of antibodies, which attach on insulin generating pancreases. The deficiency of this hormone that regulate production of glucose in our blood is result of this sugar in our blood for nutrition are not assemble and pass out of our body through urination. For this treatment stem cells can be used to replace lost or malfunctioned insulin producing cell. Stem cells can also have used to protect active beta cells that produce insulin from attack of immune system.

Alzheimer disease

It is neurodegenerative disorder. It effects brain and cause in decline of its functions such as memory, speech and though. Now a day approximately 70% of population effected by dementia is also effected with Alzheimer’s disorder. It is cause when the immune system cells microglia continue to consume arginine then those cell start to damage.

For its therapy lab experiments are continue on the lab mice in which stems cells are inoculated in brain cells of mice that can generate new connection with synapse as a result of this they will recover memory loss.

Parkinson’s diseases

It is another neurodegenerative disorder. Parkinson diseases is also term as t motion disturbs because it effect motion and equilibrium function of the brain. It may also lead to sleep disability, stress, depression and unwary attitude. It is caused due to disturbance in Dopamine production.

For its treatment old therapy was used, where stem cells are transmitted through large and compact neurons blocks. But in this method transplant neurons are also become ill. So new therapy is generated after that include inoculation of solution consist of minute amount of neuronal stem cell through arteries. These stem cells are extracted from bone marrow as result of this new therapy illness is slow down due to production of growth factors that sustenance sick.

Parkinson disease and cloning

Other useful purposes in the therapeutics is as listed below

1. Animal models can be produce through this based on human disease for the research study.
2. SCNT methods can be used to detect that specific cancer may arise either due to genetic or epigenetic disorder.
3. In genetic therapy.

Myths linked with cloning

There are some myths associate with cloning that are following

• Myth: Clones are unique animal’s DNA that are implanted onto other organism.

  Fact: not at all, they are produce like any other animal born. only main difference is that they not need any sperm and egg fusion to develop an embryo. Instead of this clone embryo are produce by using complete cell or nucleus and then inoculating it to an egg cell that is enucleated. Then embryo is grafted to uterus of female and allow to grow as in in-vitro fertilization.

• Myth: offspring of a clone is just a clone and with passage of time each generation become weaker and face more problems.

  Facts: absolutely not, they can give birth to offspring as like other animals through sexual reproduction. A breeder can use natural methods such as investor fertilization to breed clones. Thus offspring is clone but in fact resemble to offspring produce as result of sexual reproduction.

• Myth: SCNT is method that permit scientist to produce stem cells that are identical to patient thus not destroying embryo.

  Fact: SCNT is scientific method for cloning, method if successful can produce embryo and scientist use them to isolate stem cells which can destroy embryo that only few days young.

• Myth: baby produce by cloning can be grown in artificial womb

  Fact: no in real it is not possible scientist are working for fifty years to create an artificial womb but in vein. In1973, a life threating experiment was conducted on live born fetus resulted in ban on such kind of researches.

• Myth: Desire human cloning is conduct for evil purposes

  Fact: no it just simple technique uses for benefit of mankind. To create somatic cells from SCNT for repair of damaged tissues and organs and for therapeutic purposes.

• Myth: they are always physically identical to each other in looks.

  Facts: no this is not same for all. They may vary to each other in physical state such as skin margins and colors. But they may have identical genes because they consist of same set of genes but vary in their gene expression.

• Myth: life time of a clone is short span and is nearly same as of donor life span

  Fact: their birth method is same as any other animal which is sexually reproduce but it poses shorter life span compared to them but not same to donor life period. Researches are conduct in this regard for better understanding aging process.

• Myth: people produce through cloning are used as replica for spare organs of human.

  Fact: nothing could be done to a person that give him super power over normal human being. This practice is immoral and not done in any country.

• Myth: all humans clone with same genotype are rise in groups that share empathy and secret messages.

  Fact: this is totally fiction nothing else. Such kind of assumptions are just manmade and have not solid ground reasons and also against morals of laws and federal.

• Myth: cloning is used to reproduce or to bring back ancestors.

  Fact: it hardly recreates 1% of what our ancestor’s genes and only genotype not its phenotype. Because descendant has gene half from parents and half from environment thus cloning cannot develop what is from environment in our genes so it is just false assumption and not possible in reality.