In the presented case study, Mr. and Mrs. Falardeau face a dilemma regarding the future of their 3-year-old son. They want their child to be healthy and happy and doubt whether the boy would have a joyful life if he would lose his eyesight. Doing their own research, the parents consider genetic engineering as a way of treating their son’s vision. It is important to explain to the parents where genetic engineering stands these days and its prospects in treating retinal diseases.
There are positive aspects of genetic modification and gene cloning on the human body. For example, CRISPR‐Cas9, a unique technique of genetic engineering discovered in the 1990s, can find specific pieces of DNA that need to be modified and do so (Xu et al., 2018). CRISPR‐Cas9 is found to be “efficacious and cost-effective” in treating retinal diseases such as retinitis pigmentosa, which is caused by the loss of photoreceptor cells that can cause blindness (Xu et al., 2018, p. 26). Ethically, research shows that it would be impermissible not to cure people using something as easy as modifying genes (Liao, 2019). However, it is crucial to consider that interfering with genes can have irreparable consequences.
The negative aspects of cloning and genetic modification are hard to foretell because there is relatively little research on humans. Modifying human genes may result in inheritable changes that cause various risks, whereas cloning may have unpredictable effects on future generations (Shafique, 2020). However, there are techniques of genetic engineering, such as CRISPR‐Cas9, that with every day become more promising. In 2020, CRISPR‐Cas9 was used in treating patients with cancer, and the results show no “off‐target effects and clinical toxicities” associated with CRISPR-Cas9 (Zhang, 2021, p. 2476). With each person’s health being unique, there can be many outcomes that can result in life expectancy differently, but there are more hopeful prospects every day.
While CRISPR‐Cas9 is being studied to treat a wide range of diseases, it has already shown positive effects in treating retinal diseases without life-threatening consequences. It would be cruel not to give a chance of treatment to a child who would lose his vision before he turns eight years old when children only begin to truly explore the world. Even though it is hard to predict all the outcomes of genetic modification and cloning, I would suggest using CRISPR‐Cas9 in treating retinal diseases such as the one described in the case study.
References
Liao, S. M. (2019). Designing humans: A human rights approach. Bioethics, 33(1), 98-104.
Shafique, S. (2020). Scientific and ethical implications of human and animal cloning. International Journal of Science, Technology and Society, 8(1), 9-17.
Xu, C. L., Park, K. S., & Tsang, S. H. (2018). CRISPR/Cas9 genome surgery for retinal diseases. Drug Discovery Today: Technologies, 28, 23-32.
Zhang, B. (2021). CRISPR/Cas gene therapy. Journal of Cellular Physiology, 236(4), 2459-2481.
Cloning is a biological process of producing populations with genetically identical DNA as their predecessors through asexual reproduction. In layman’s terms, it’s like producing multiple copies of a product that are identical to the original/parent product. My essay will focus on reproductive cloning which refers to a procedure of creating new multi-cellular organisms that are genetically identical to each other. Fertilization of the eggs or gametes from the hosts does not occur hence the mode of reproduction is asexual. Asexual reproduction is however a “naturally occurring phenomenon in many plant species through vegetative reproduction” (Panno, 2004, p. 57) and other species like an amoeba. Reproductive cloning is a very controversial issue in today’s society with a majority of people fearing scientists are playing God and they may attempt to clone humans. Currently, most of the attempts involve the cloning of certain animal species like Dolly the sheep, and only a few of these attempts have been successful. Reproductive cloning utilizes a procedure called “somatic cell transfer to come up with offspring that are genetically identical to the parent nucleus” (Panno, 2004, p. 61). This process entails the removal of a nucleus from a host’s cell and transferring it to an egg that lacks a nucleus. It is the nucleus that contains the DNA of the donor. The nucleus undergoes reprogramming once it is inserted into the egg. (Reprogramming is the removal and remodeling of epigenetic marks) The egg which now contains the nucleus (and DNA) of the donor is stimulated by an electric shock and starts dividing. After a couple of mitotic cell divisions, an early-stage embryo known as a blastocyst is formed. Its DNA structure is similar to the donor. “The blastocyst is then transferred to the uterus of a surrogate mother”. (Panno, 2004, p. 63).
Injaz the Cloned Camel
Another prominent example of animal cloning was Injaz; the world’s first cloned camel. Injaz was cloned in Dubai, United Arab Emirates. Injaz was “created” from the collected ovarian cells of a donor camel that had been killed for its meat in 2005. The cells were then grown separate from an organism in tissue culture and then preserved in liquid nitrogen. Later, one of the cells was transferred into the egg or oocyte of a surrogate camel whose nucleus had been removed. An electric shock was passed to join them and chemically stimulated to kick off cell division. Sub-division of the cells to form a blastocyst continued for a week after which the embryo was implanted into the surrogate camel’s uterus. “The procedure was successful and pregnancy was confirmed twenty-eight days later using an ultrasound” (2009). The gestation period lasted 378 days after which Injaz was born. Injaz was proven to be a clone of the original camel when both DNAs were found to be similar after being tested at the Molecular Biology and Genetics Laboratory in Dubai. “According to Dr. Lulu Skidmore camel cloning gave them the means to preserve the valuable genetics of their elite racing and milk-producing camels of the future.” It’s worth noting that camel racing is a lucrative industry in the Arab world and this is seen by the “personal endorsement and support for the project by the vice president of the UAE and the emir of Dubai” (2009).
Summary
Animal cloning is certainly a breakthrough especially in the field of medicinal science where experts believe the cure of diseases like diabetes and other genetically inherited diseases can be found if similar cloning procedures can be allowed in stem cell research. This will certainly be useful to mankind if and only if strict regulation is introduced to monitor scientific research. The chance of a human clone occurring is a question of when and not if. (Panno, 2004, p. 111).
References
Panno Joseph PhD, (2004) Animal Cloning: The Science of Nuclear Transfer, Facts on File, pp. 56-63
Panno Joseph PhD, (2004) Gene Therapy: Treating Disease by Repairing Genes, Facts on File, pp. 109-113
USA Today, 2009, Scientist: First Cloned Camel Born in Dubai.
In the last century, humanity was confronted with such an outstanding scientific achievement as cloning. In 1966, the world was struck by the successful cloning of a sheep known as Dolly the sheep. Soon after that, many scientists began their research on cloning various animals, as well as humans. However, this scientific interest was quickly halted due to bans imposed by many nations that found the process unethical and violated human rights. Until now, the scientific community has banned cloning or that living organism, and I believe this is the right decision for several reasons.
Many scientists have seen cloning as an opportunity to revive extinct species. However, I do not consider this a necessary endeavor. In my opinion, the revival of extinct species such as mammoths will not lead to anything good but instead to disaster. One can hardly imagine giant creatures like mammoths walking around in the streets instead of the pets we are used to. Such interference in the process of natural selection will only lead to the loss of biodiversity because animals like mammoths will need habitat, a suitable ecosystem, and food. In addition, knowing the selfishness of people, the appearance of such rare animals will lead to an even more significant development of poaching and the popularity of the black market. Fortunately, scientists have concluded that it is almost impossible to revive such ancient animals as mammoths because of the problems with DNA, which will be challenging to recover from the remains of many years. Since they have been subjected to destruction from cold temperatures, scientists have concluded that DNA is a fragile molecule that does not stay intact for long after death.
Scientists also wanted to use the cloning method to grow organs for transplantation. I agree that this idea can be beneficial and works for the good of humanity. However, increasing organs by cloning requires embryonic cells for study, which is unethical. After all, an embryo is recognized as a living organism, and for cloning experiments, embryo cells would have to be killed in the research. It violates all human rights since no one has the right to take someone’s life.
Moreover, this method is not the only one, and there are alternatives that are more ethical and effective ways, such as growing organs from stem cells. Xenotransplantation, or transplanting animal organs into humans, is another method. Therefore, these technological advances indicate that cloning might not be necessary to harness those valuable cells.
Thus, I believe that cloning should be prohibited. After all, this process will lead to biodiversity loss and violate all ethical norms and principles.
The cloning debate has been going on for a while now. Whether cloning is good or bad is yet to be generally agreed upon because people have different opinions. According to Baird, human cloning should be prohibited for the simple reason that the onus of justification will be placed on the shoulders of those performing the cloning rather than those who want the cloning done. Baird’s implications are, however, misplaced because we need a good reason to limit the actions of others, especially when those actions are not bringing any harm to anyone, for example, an activity should not be forbidden just because a portion of the society finds it distasteful even if it does not bring any harm to them.
Many critics have argued that human cloning is harmful to the clones even though there has not been any formally documented evidence of the harm of cloning. Human cloning, especially for reproductive purposes, has been increasingly practiced and also criticized. It should be well tested and attempts made on animals before trying it on human beings to determine whether it has any effects that are negative.
Baird is still against human reproductive cloning because she believes that it might change the way society views children. She argues further that if children are cloned, then they will be viewed as a commodity because of the expensive nature of cloning. Other researchers have, however, revoked these claims by stating that it is also costly to raise normal children from childhood to adulthood, and therefore the costs of cloning should not worry us or make us love the cloned children any less.
Baird still argues against cloning that it does not support human identity and where they come from, for example, the sense of arising from a maternal or paternal line. We can however, discard these arguments by stating that most normal people born normally do not identify with where they come from either their maternal or paternal lines for example someone who is raised by a single mother identifies only with the maternal heritage.
This means that dual heritage is normal and desired but it does not affect our conception as human beings. Baird continues to argue that we should also look at the social effects of cloning rather than the individual effects only. He also argues further that cloning is relatively a new technology and should be carefully reviewed before it is allowed. This is reasonable but it does not mean that cloning should be totally banned as Baird and other critics suggest. They should instead champion for discussion and regulation on cloning for example limiting the number of clones that might be created from one individual.
Bayis in her article on cloning argues that cloning should be thought of as an enhancement on technology and not a reproductive technology and that this way it might be easily accepted. As mush as cloning should be accepted, it does not mean that it is totally good or that it has no negative effects. This is because cloning is not right in all circumstances. Some people carry out human cloning for malicious reasons.
This means that cloning is only appropriate in a few cases and therefore caution should be taken towards cloning. The public should also not direct all its resources towards cloning because sometimes cloning is bad. A ban on research activities that lead towards human cloning is not necessary even though there should be caution and discretion towards cloning.
Molecular cloning is a set of methods in molecular biology that is used to obtain multiple copies of the target DNA fragment. This procedure includes several steps: preparation of cloning vector and a target DNA fragment, preparation of a recombinant DNA, and its insertion into a host organism. Target DNA fragment – it is usually a coding gene or a regulatory element. For molecular cloning procedures, it should be isolated, amplified, and purified. As a vector, bacterial plasmids and bacteriophages are used.
Bacterial transformation is a process of recombinant DNA (a cloning vector linked with a target DNA fragment) insertion into a host bacterial cells (1). Various methods could be used for bacterial transformation with plasmid vectors, including chemical treatment of cells with salts (in particular, CaCl2), heating the bacterial suspension, or electroporation. All the approaches have the same principle: after the treatment, small holes appear in the bacterial membrane, which makes the process of plasmid uptake easier (2).
The electroporation technique is widely used for bacterial cell transformation. It was stated that the efficiency of this process is up to 80% of survived transformed cells (3). For this procedure, the suspension of bacterial cells and recombinant DNA plasmids are subjected to brief electrical impulses. It results in small holes that appear in bacterial membranes. Plasmids could easily penetrate into bacterial cytosol through these holes (4).
After plasmid insertion, it is required to select transformed cells. Several approaches are used for this procedure. A recombinant vector contains genes that allow the identification and selection of transformed cells. It could be a gene of certain antibiotic resistance (1) or a Green Fluorescent Protein (GFP) gene. GFP is a protein from jellyfish Aequorea Victoria. It is responsible for glowing points of light around the margin of its umbrella (5).
In response to ultra-violet radiation, GFP produces green fluorescence emission. According to the technique, the GFP-coding gene introduced downstream of the promoter region of a target gene. Therefore, if cells produce GFP, they also produce target molecules. GFP gene expression can be easily detected under the ultra-violet radiation. Bacterial colonies that produce green fluorescence could be visually identified and isolated (6).
GFP plasmids (pGFPs) are widely used in molecular cloning techniques in biomedical researches. It is used for human cells transformation in cancer studies (7), animals (8), and bacterial (9) cells transformation. In biotechnology, molecular cloning with pGFP is widely used to insert genes of target protein production (enzymes, human antibodies, recombinant medicaments such as antibiotics and insulin, protein for vaccines production, and others) (10).
In this laboratory report, the process of bacterial cell transformation is described. This project is dedicated to recombinant plasmid construction and bacterial cells transformation. It includes several steps:
Growing Escherichia coli bacteria with a plasmid with a gene of ampicillin resistance (pGFP) and a plasmid with a gene of Chloramphenicol resistance (pBCKS) and plasmids isolation.
GFP gene from pGFP restriction and its insertion into pBCKS.
Bacterial cells with recombinant pBCKS transformation.
Transformed cells detection and selection.
Restriction enzymes treatment is used to isolate GFP fragment from the ampicillin vector. In the Chloramphenicol vector, GFP fragment occurs behind the Lac polymerase promoter region. In this case the plasmid sequence orientation and GFP gene expression are correct. The final step of the project is bacterial selection. Successfully transformed bacteria cells will be Chloramphenicol resistant and will produce green fluorescence emission with the UV light.
Aim
The aim of this project was to insert Green Fluorescent Protein (GFP) coding gene into pBSCK vector and to introduce this vector into E. coli bacteria cells.
Materials and Methods
All materials and procedures were explained in “ONPS1052” RMIT Gene Technologies 1 Practical notes 2017 by Mouradov (2).
Results
In the study, pGFP and pBCKS plasmids were used. Figure 1 shows the structure of plasmids.
Figure 1. Plasmid map of pGFP and pBCKS used in this study.
Both plasmids have the similar structure. The pGFP vector contains a selectable marker (the ampicillin resistance gene) and a visual marker (GFP). The pBCKS contains chloramphenicol resistance gene as a selectable marker. Both plasmids contained multiple cloning sites (MCS) with restriction sites for EcoRI and HindIII enzymes (2).These plasmids were inserted into competent E. coli strains. Liquid LB cultural media with ampicillin and chloramphenicol antibiotics were used for growing transformed E. coli. Bacteria cells numbers are shown in Table 1.
Table 1. E. coli with pGFP and pBCSK plasmids colonies count on LB medium containing ampicillin or chloramphenicol correspondingly.
Plate (plasmid)
Bacteria cells count
per 10 µl
per 100 µl
Ampicillin (pGFP)
45
456
Chloramphenicol (pBCKS)
20
289
The high number of transformed single E.coli colonies containing pGFP plasmid and pBCKS plasmids grow on LB medium containing either ampicillin antibiotic (pGFP ampicillin resistance) or Chloramphenicol antibiotic (pBCKS Chloramphenicol resistance). It could be stated that pGFP transformation was more efficient because the concentration of transformed cells with pGFP per 10 µl of a suspension was more than twice higher, and per 100 µl was around 1.6 times higher in comparison to pBCKS transformed cells’ concentration. The difference between 10 µl and 100 µl results could be considered as insignificant. In general, both processes of transformation were successful, and cells with target plasmids were obtained.
Extracted plasmids treated and non-treated with EcoRI and HindIII enzymes were analyzed by electrophoresis in agarose gel. The results are presented in figure 2. According to the results, digested pGFP contained non-restricted GFP plasmid (~2600bp) and a fragment of GFP gene (~800bp). Digested pBCKS contained non-restricted plasmid (~3100bp).
Figure 2. Extracted plasmids treated and non-treated with EcoRI and HindIII enzymes.
Group results (1-4 lines) of electrophoresis in agarose gel of extracted plasmids treated by digestive enzymes (EcoRI + HindIII) as well as non-treated plasmids. L= Ladder; lane number 1= pGFP; lane number 2= pBCKS; lane number 3= Digested pGFP; lane number 4= Digested pBCKS.
The procedure of ligation of two restricted plasmids was conducted. As a result, a new recombinant pBCKS plasmid contained BFP gene was expected to appear. On the last stage of the project, competent E. coli cells were transformed with the recombinant plasmid (ligation of the GFP with pBCKS backbone). 10 and 100 µl of transformed bacteria mixture were cultivated on a solid LB medium with chloramphenicol (Ch.) and isopropyl β-D-1-thiogalactopyranoside (IPTG) (fig. 3).
Only cells with introduced recombinant pBCKS plasmid were able to grow on this media. Chloramphenicol was used to select bacteria with a gene of resistance to this antibiotic which was a part of pBCKS. IPTG is a compound which induced the expression of genes which are controlled by Lac promoter. Therefore, only in presence of this compound, plasmid genes expression is possible (11).
Figure 3. Growth result of 10 µl and 100 µl of E. coli transformed with a ligation product on LB plates with chloramphenicol and IPTG.
The presence of GFP gene was detected under the UV light. Under this condition, GFP started to immerse green fluorescence which could be easily detected by the naked eye (fig. 4).
Figure 4. Growth result of 10 µl and 100 µl of E coli transformed with a ligation product on LB plates with chloramphenicol and IPTG under UV light.
Discussion
The initial transformation of E. coli using both types of plasmids, shown in Figure 1, was successfully conducted. The high number of single E.coli colonies was obtained on solid LB medium contained either ampicillin or chloramphenicol antibiotics as selection factors for E. coli containing pGFP and pBCKS plasmids, correspondingly. The results are shown in Table 1. After culturing of a few colonies in LB broth and incubating overnight, plasmids were then extracted from transformed E. coli cells and digested with HindIII and EcoRI restriction enzymes. The digested and non-digested plasmids were run on the electrophoresis in agarose gel. Results are shown in Figure 2.
In case of non-digested plasmids, pGFP plasmid (the lane number 1, Figure 2), two bands were obtained, while it was expected o get one band around 3.3Kb. For non-digested pBCKS plasmid (the lane number 2, Figure 2), results were the same. In case of digested pGFP plasmid in the lane number 3 (Figure 2), two bands around 2600bp and 800bp were observed. In the related to digested pBCKS lane number 4 (Figure 2), one band slightly over 3100bp was observed.
After completing a sub-cloning stage, 10 µl and 100 µl of transformed E. coli contained recombinant pBCKS plasmids were cultured on an LB plate with chloramphenicol and IPTG (Figure 3). After cultivation of 10 µl mixture, 43 single E. coli colonies were observed on a plate with selective medium. After cultivation of 100 µl, approximately 250 single E. coli colonies were observed. The green fluorescence emission was detected under UV light, as it is shown in Figure 4.
In this study, it was confirmed that the plasmids could be successfully extracted from transformed E. coli. More than one 3300bp and 3400bp bands were observed on the gel for extracted pGFP and pBCKS plasmids, respectively. This could be explained by the fact that plasmid DNA can exist in three conformations on the gel: supercoiled, open circular, and linear. Linear DNA migrates with the slowest speed, in comparison to other forms (12). Therefore, the lowest observed on the gel bands (Figure 2) are assumed to be supercoiled form of non-digested pGFP and PBCKS plasmids. In case of digested pGFP plasmid, GFP band was detected at 800bp as it was expected. It means that the target GFP gene fragment has been successfully restricted by enzymes (HindIII and EcoRI).
After sub-cloning the GFP fragment to pBCKS plasmids and cultivation E. coli on an LB plate contained chloramphenicol and IPTG, the correct orientation of GFP sequence behind the lac promoter sequence was examined by screening emission of green fluorescence from E. coli colonies under the UV light. Interestingly, high number of green fluorescence emission E. coli was observed on the selected media. It means that the ligation was conducted efficiently with the high rate of recombinant plasmid formation. After that, the process of transformation was also effective. Therefore, a high number of E. coli colonies with plasmids contained both chloramphenicol resistance genes and GFP genes was obtained in the study.
In general, this project demonstrated the efficiency of protocols of plasmids isolation, restriction, and legation and transformation of competent E. coli cells with recombinant plasmid. Effective methods of detection (growing on selective media contained antibiotics and visualization of green fluorescence under UB light) were tested. Besides, the effectiveness of plasmid restriction was tested using the electrophoresis in agarose gel.
References
Rapley R, Whitehouse D, editors. Molecular biology and biotechnology. London: Royal Society of Chemistry; 2014.
Mouradov A. RMIT gen technologies practical notes, 1st semester. Melbourne: School of Applied Sciences, RMIT University; 2017.
Jordan CA, Neumann E, Sowers AE, editors. Electroporation and electrofusion in cell biology. Berlin: Springer Science & Business Media; 2013
Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation. Nucl. Acids Res, 1988; 16: 6127-6145.
Shimomura O, Johnson FH, Saiga Y, Cell J. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. Journal of Cellular and Comparative Physiology, 1962; 59: 223-239.
Cseke LJ, Kirakosyan A, Kaufman PB, Westfall MV, editors. Handbook of molecular and cellular methods in biology and medicine. 3rd ed. Boca Raton: CRC Press; 2016.
Gao J, Chang MT, Johnsen HC, Gao SP, Sylvester BE, Sumer SO, et al. (2017). 3D clusters of somatic mutations in cancer reveal numerous rare mutations as functional targets. Genome Medicine, 2017; 9(1): 4-17.
Karunakaran DKP, Kanadia R. In vivo and explant electroporation of morpholinos in the developing mouse retina. Morpholino Oligomers: Methods and Protocols, 2017; 1565: 215-227.
Baty AM, Eastburn CC, Diwu Z, Techkarnjanaruk S, Goodman AE, Geesey GG. Differentiation of chitinase-active and non-chitinase-active subpopulations of a marine bacterium during chitin degradation. Appl. Environ. Microbiol., 2000; 66: 3566–3573.
Lipps G. Plasmids: current research and future trends. New York: Horizon Scientific Press; 2008.
Gene technologies. Practical manual. Melbourne: School of Applied Sciences, RMIT University; 2015.
Swamy PPM. Laboratory manual on biotechnology. New Delhi: Rastogi Publications; 2008.
Cloning at its basics is the use of artificial genetic and reproduction processes to create a genetically identical copy of any biological entity or material (cells, tissues, or genes). For animal or human reproductive cloning, the process is complex. A mature somatic cell, usually a skin cell, is removed and placed into an egg cell (oocyte) where the nucleus has been removed. This can be done directly via a needle or an electric shock to fuse the two. The egg then develops in an early-stage embryo before being placed in the womb of an adult female animal as a surrogate. The clone develops in the womb and eventually, the adult female gives birth, with the new clone having an identical genetic makeup to the organism from which the somatic cell originated (National Human Genome Research Institute, 2021).
The largest breakthrough in cloning occurred in 1997 when scientists in Scotland cloned a sheep Dolly via somatic cell nuclear transfer, also known as reproductive cloning, as described above. Häyry (2018) examines the ethical issues surrounding cloning in his comprehensive article. Immediately after the 1997 event, most countries around the world and international organizations banned cloning. However, the ban does not apply to animals as scientists have cloned several animals since, nor does it apply to the technique of somatic cell nuclear transfer which is actively used in in-vitro fertilization (IVF) to increase odds of success.
Therefore, the conjecture is that the combination of human cloning using somatic cell nuclear transfer is the primary issue at hand. Virtually all countries prohibit human cloning, while a small minority allows for therapeutic cloning and embryonic stem cell research which may use nuclear transfer, but any cloning done on human somatic cells is not permitted to reach a viable state.
Häyry (2018) identifies three primary ethical issues with human cloning. First is the possibility of asexual reproduction which could lead to distorted families. Experts argue that humanity’s continuous renewal depends on heterosexual reproduction, it allows for the creation and evolution of the human genome as well as the numerous social benefits of families and connections. Asexual organisms are commonly selfish with only a single goal in mind which is to pass their genome as a whole.
The next ethical concern is elements of design, control, and deformed societies. Cloning could destroy contemporary society – if, given the option of human cloning, parents or governments will want control over the reproductive process, which will lead to the destruction of the value of human life, self-awareness and freedom, and the unspoken truth that no human is the ‘maker’ of another. Finally, there is the universal ethical concern that cloning could be used to genetically enhance and customize human beings. Creating enhanced human beings, will lead to the stratification of society based on the genome as well as eliminate many things that make us human, which are flaws, imperfections, and weakness to mortality.
If given a choice to clone a loved one or a favorite animal, especially if they have passed, it is a significant emotional and ethical dilemma. First, to emphasize, I am against cloning living individuals or even personal animals. The only potential for cloning living animals is perhaps something like livestock to generate more food sources. As for an opportunity to create a clone of a deceased creature, to have the potential to see them again and spend time together, I would argue against it. Unfortunately, a clone is simply a physical body, missing the unique characteristics of a person such as personality, intellect, and emotional quotient.
It is unlikely with any realistic technology that scientists could transfer personality across bodies. However, if such technology does get developed at one point, one cannot transfer the ‘soul’ of an individual if one believes in that, and it is also impossible to mimic the upbringing of the person. Therefore, in one way or another, the person will be different from their original self, which would be highly unnatural and abnormal.
References
Häyry, M. (2018). Ethics and cloning. British Medical Bulletin, 128(1), 15–21. Web.
National Human Genome Research Institute. (2021). Cloning fact sheet. Web.
When two or more genes that have different kinds of gene code are combined they form Chimeric or fusion proteins. These genes are coded for separate proteins. Eventually a new polypeptide is formed with the functional properties of the proteins that were combined (Tierney, 2007).
DNA cloning can either be based on cells or achieved by using polymerase chain reaction(PCR).In the approach based on cells both the replicating molecule or the biological vehicle known as the vector and the foreign DNA fragment are cut using the same restriction enzyme(s ) to produce compatible cohesive (“sticky”) or blunt ends on the DNA molecules, then the foreign DNA fragment is permanently joined to the DNA of the vector using an enzyme known as DNA ligase which catalyzes the formation of a phosphodiester bond between the two DNA chains thus producing a chimera or a recombinant DNA molecule (Rychlik, 1990). The replicating molecule is meant to carry the foreign DNA fragment into the host cell (Chou & Bloch, 1992).
Fusion proteins can occur naturally by a process known as chimeric mutation. This process occurs when there is a high level of mutation. This creates a new coding system from the two genes.
Fusion proteins can be created in two ways. There is the artificial way and this is known as Recombinant fusion. This kind of fusion is got from the process known as recombinant DNA technology. This technology is used for research in biological fields.
Steps
DNA cloning involves the following steps;
DNA recombination-This involves the identification and isolation of the DNA fragment containing the gene of interest from the chromosomal DNA using restriction enzymes or by using the polymerase chain reaction(PCR),gel electrophoresis and sonication of DNA (Tierney, 2007). The fragment of DNA isolated must be joined to a replicating molecule or vector which acts as a vehicle that transports the DNA into the host cell. Both the isolated DNA fragment and the vector are cut using restriction enzymes at their restriction sites into sizeable fragments suitable for cloning. The desired DNA fragment is inserted into the cut ends of the vector and permanently linked using the enzyme DNA ligase thus forming a recombinant DNA molecule or chimera, but in some instances if processed under in vivo conditions, the enzyme terminal transferase may be added in order to avoid free sticky ends to rejoin instead of forming a chimera since it catalyzes the addition of “tails” of the nucleotide to the 3’ends of the DNA chains.
Transformation-This is whereby the recombinant DNA molecule enters the host cell (which is usually a bacterium) and proliferates. The recombinant plasmid molecule also contains color selection markers which show white/blue screening on a media of X-gal (Saiki & Arnheim, 1985).
Selective amplification-Within the host bacterium the vector multiplies producing numerous identical copies not only of itself but also of the gene that it carries. After a large number of cell divisions, a colony or a clone of identical host cells is produced.
Isolation of desired DNA clones-Culturing of transfected cells is done. The selectable antibiotic resistance markers are used as well as the color selection markers if present in the recombinant plasmid, though further confirmation.
This paper aims to discuss how the fusion proteins are formed from the two processes.
Recombinant Fusion Protein
The first step in this process is identification of the regions of the DNA that are going to be used in the synthesis. This is done by use of bioinformatics. When these regions are synthesized they form antigenic fragments. The region that binds to the antigen on an antibody is known as the fragment. It has a single constant and variable domain of the chain (Talpaz, 2003).
The fragments can be designed for a specific region if required (Perrone, 2005).
The second step is the design of polymerase chain reaction primers. The primers are used to amplify these regions. Primers are used as the starting point to synthesis of DNA. The polymerase chain reaction is used to magnify DNA strands through several orders of magnitude. This leads to the replication of the DNA. This process consists of a subsequent repetition of 20-40 temperature changes. These steps known cycles consist of 2-3 discrete temperature steps. This process happens in three stages:
Exponential amplification: This involves doubling the product in every cycle.
Leveling off stage: This involves slowing down of the reaction until the DNA polymerase loses activity.
Plateau: There is no more formation of products as the entire reagent and enzyme for the reaction is used up (McFarland, 2009).
The third step involves cloning of the DNA using different expression systems. The regions of the DNA that were amplified are first purified using a gel in preparation for the cloning stage. They are then isolated and after that screened for expression performance (Perrone, 2005).
Sequencing and Alignment Analysis is the fourth step in this process. The DNA is arranged in such a way that the regions will be similar in function and structure.
The fifth step is to identify the clones that are relevant for the synthesis. This is can be done in two ways depending on the expression yields required (Kitcher, 2007).
The two are Higher-plex techniques and Low-to-mid-plex techniques.
After that the conditions and additives are optimised so as to improve on the yield of the clones.( Othman& Hart, 1988).
Lastly purification is done to the clones so as to get the required yield. The isolated DNA is purified and placed on electrophoretic gel. Purification is done to produce a yield of more than 94% in purity (McLaren, 2000). Purification of the DNA fragments is important to ensure the integrity of the results. Consequently, the presence of these fragments may affect the clarity and the precision of the results.
Chimeric mutation
The other way that fusion proteins can be formed is through chimeric mutation. The process in which this mutation occurs is known as chromosome translocation. This is a naturally occurring process caused by non homologous chromosomes reconfiguring themselves (Khan & Park, 2008). A fusion is then formed when the two or more genes combine. This is most profound in cancer. This occurrence can be detected by cytogenetic of the cells affected. Translocations can either be balanced or unbalanced. When balanced there is exchange that is in consequential to the gene code but for unbalanced it leads to the missing of some genes thus a chromosome imbalance (Nekton, 1989).
There are two types of translocations (Liu, 1995).
Reciprocal (non Robertsonian) this translocation occurs when there is exchange of materials between chromosomes that are non homologous. This kind of translocation is not harmful. This is normally found in prenatal diagnosis. At times carries of balanced translocations react with those unbalanced translocations. This may lead to miscarriages or abnormality in newborns (Kitcher, 2007).
Robertsonian translocation involves the combining of two or more chromosomes. This happens at the centromere region which leads to the loss of a short arm. The chromosomes are acrocentric. The rearrangement results in a karyotype. Newborns have a have translocations. They occur at chromosomes 13 and 14 (Vicente, 2004).
Translocations are known to cause several diseases in human beings:
Cancer
Infertility
Down syndrome
Conclusion
This paper has so far analyzed the making of fusion tags and their future use in our day to day lives. Fusion tags can be used in different applications, thus enhancing and benefiting the way bodily processes are carried out (Higuchi, 1988).
References
Chou, M. & W. Bloch (1992). Prevention of pre- PCR mis-priming and primer dimerization improves low-copy-number amplifications. Japan: Hanoi publishers.
Higuchi, R. (1988). Primer-directed enzymatic amplification of DNA with a Thermo stable DNA polymerase. Japan: Longhorn publishers.
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Pectate lyase is an enzyme involved in the catabolism of pectate in cell walls of plants. Dubey et al. (2016) describe pectate as de-esterified pectin forming the main component of carbohydrates in the cell wall. Classification indicates that pectate lyase falls in the family of lyases, which cleave oxygen-carbon bonds in polysaccharides. Plant pathogens, such as yeast and bacteria, as well as symbiotic bacteria in the gut of ruminants, use pectate lyase in degrading pectate and deriving energy they need.
Hugouvieux-Cotte-Pattat et al. (2014) explain that pectate lyase applies the mechanism of β-elimination in degrading pectate to short polysaccharides comprising of 4-deoxy-α-D-galact-4-enuronosyl groups. In plant pathogenesis, pectate lyases contribute to the degradation of polysaccharides into oligosaccharides for other enzymes to continue with the degradation. Therefore, the understanding of structure and functions of pectate lyases plays a central role in their applications in industrial uses because they help in the catabolism of polysaccharides in cell walls, such as pectin and pectate. The emergence of molecular cloning has enhanced the application of pectate lyases in industrial processes of manufacturing natural fibres and fruit juices.
Pectate lyases are highly diverse because they vary according to their sources, structure and functions. Pectate lyase 1 (Pel1) is one of the established pectate lyases that has been created as a recombinant protein. The size of Pel1 gene is 1080bp that codes for an enzyme with 360 amino acid sequences. Cloning of this gene into an expression vector allows expression of its protein. Gay et al. (2014) recommend the use of expression vectors that are small and easy to clone, isolate and purify.
Therefore, the objectives of the series of experiments performed were to extract genomic DNA from Northumbrium northumbria, amplify Pel1 gene, clone it into the expression vector (pET-28a) and transform BL21 cells. Subsequently, the experiment aimed to express, purify, crystallise and characterise the recombinant Pel1. The overall objective of these experiments was to produce a recombinant Pel1 and determine its 3-D structure using x-crystallography.
Results
Extracted Genomic DNA
Figure 1 shows a clear band (4) of isolated genomic DNA from Northumbium northumbria.
Figure 1: Bands of Bioline Hyperladder I (L) and genomic band of isolated DNA (4).
PCR Product
Figure 2 depicts results of PCR amplification of Pel1 gene resolving at the correct positions relative to the ladder.
Figure 2: Bands of Bioline Hyperladder I (L) and band (4) of PCR product of Pel1 gene (1080bp).
Plasmid Purification
Figure 3 indicates bands of transformed and purified plasmids of Escherichia coli (BL21).
Figure 3: Agarose gel photo of purified plasmid bands with the well number 4 without expected band due to human error.
Concentration of Pel1 (ug/ul)
Absorbance = 0.055
Molar Absorptivity Coefficient (Ԑ) = 84800
Concentration (C)
Length (L) = 1
Relative molecular mass = 43203
A = ԐCL
Therefore, C = 0.055/84800L/mol*cm*1cm = 6.486⨉10-7 mol/L
Figure 6 is an image of plate-shaped crystals of Pel1 obtained from the expression of the gene in Escherichia coli.
Figure 6: Crystallised protein (Pel1)
Discussion
The objective of isolating genomic DNA from Northumbrium northumbria was achieved successful due to the formation of a clear band at the expected genomic size that is more than 10kbp (Figure 1). According to Mulcahy et al. (2016), a sharp band without smears indicates that an isolated genomic DNA is not also pure but also quality. The objective of amplification was achieved effectively because PCR generated the expected product of 1080bp, which resolved correctly in agarose gel (Figure 2).
The cloning of PCR products into the expression vector (pET-28a) was performed successfully. Restriction digests by enzymes (Xho I and Nde I) enabled ligation of Pel1 gene into the expression vector. Transformation of the E. coli (BL21) with the plasmid cloned with Pel1 occurred effectively. However, purification of plasmids did not materialise due to the loss of sample during purification.
Despite the occurrence of a human error during purification of plasmids, the expression of Pel1 was fruitful. Gay et al. (2014) hold that successful cloning and expression of the gene of interest is dependent on the size of an insert, the nature of a selectable marker and the presence of control elements. Based on the Beer-Lambert’s law, the molar absorptivity coefficient of 84800 L/mol.com and relative molecular mass of 43203, the concentration of Pel1 is 0.28ug/ul. Robinson (2015) explains that the determination of the concentration of enzyme forms the basis of calculating its activity. The SDS-PAGE reveals that the size of the expressed protein is about 15kD and crystal analysis shows that the size is 4kD.
In contrast, the expected theoretical size of Pel1 is approximately 35kDa. Guan et al. (2015) assert that systemic errors and modification of amino acid sequences explain differences in theoretical and experimental molecular weights of proteins. The activity rate of Pel1 is 8.82*10-8 Unit/ml, which is very low when compared to literature value of between 5.47 and 43.53 Unit/ml (Muslim et al., 2015). Eventually, crystallisation of Pel1 led to the successful production of plate-shaped crystals.
References
Dubey AK, Yadav S, Kumar M, Anand G and Yadav D (2016) Molecular biology of microbial pectate lyase: a review. British Biotechnology Journal 13, 1-26.
Gay G, Wagner DT, Keatinge-Clay AT and Gay DC (2014) Rapid modification of the pET-28 expression vector for ligation independent cloning using homologous recombination in Saccharomyces cerevisiae. Plasmid 76, 66-71.
Guan Y, Zhu Q, Huang D, Zhao S, Lo LJ and Peng J (2015) An equation to estimate the difference between theoretically predicted and SDS PAGE-displayed molecular weights for an acidic peptide. Scientific Reports 5, 1-11.
Hugouvieux-Cotte-Pattat N, Condemine G and Shevchik VE (2014) Bacterial pectate lyases, structural and functional diversity. Environmental Microbiology Reports 6, 427-440.
Mulcahy DG, Macdonald KS, Brady SG, Meyer C, Barker KB and Coddington J (2016) Greater than X kb: a quantitative assessment of preservation conditions on genomic DNA quality and a proposed standard for genome-quality. DNAPeerJ 4, 1-12.
Muslim SN, Al-Kademy IMS, Mahammed AN, Musafer HK and Muslim, SN (2015) Detection of the optimal conditions for pectate lyase productivity and activity by Erwinia chrysanthemi. Journal of Medical and Bioengineering 4, 184-191.
Robinson PK (2015) Enzymes: principles and biotechnological applications. Essays in Biochemistry 59, 1-41.
Ever since the beginning, mankind has been besieged by disease. Many of these diseases have been healed by scientists in their respective laboratories, but there are still many diseases that have escaped solution: Diabetes, Heart disease, Parkinsons disease, Lou Gehrigs’ disease and many other illnesses and devastating disabilities. A promising avenue for research that might lead to possible cures is the ESC or the Embryonic Stem Cell. Critics point this out, but also acknowledge that such research destroys embryos and that pursuing such means would show insufficient regard for human life. Since human embryos are morally important, it follows that substantial limits must be imposed on continued research.
Those who oppose such research take into consideration its potential and the possibility of its one day yielding substantial medical benefits. They do not lose sight of the fact that there are other chances for progress in basic research and for developing models to study different diseases. Recent results in research involving non-embryonic and adult stem cells point out that scientists may be able to make progress in regenerative medicine without resorting to cloning for biomedical research.
There is in existence the so-called “Nuremberg Code of Research Ethics” which enunciates the principle that experimentation should be “such as to yield fruitful results for the good of society, unprocurable by other methods or means of study.” Unfortunately, because of the bulk of scientific uncertainties and the possible avenues of research, the problem at present evades solution. If we contemplate the fact that research using cloned embryos may yield knowledge and benefits that cannot be derived from any other means, what reasons could we have for saying “no” – for turning down cloning for biomedical research?
Since no one can possibly know or predict for certain which avenues of research will prove most successful – not the scholars, not the moralists, not the patients whose suffering we all hope to alleviate – it would be wise to leave this possible avenue to medical progress open. This approach forces us to think about embryo research generally; however cloning, even only for purposes of research raises its own concerns, since only cloned embryos couls one day become cloned children.
The analysis of those against cloning for biomedical research proceeds along three pathways; what we owe to the embryo; what we owe to society; and what we owe to the suffering. They differ among themselves, or the relative importance of the various arguments presented. But they all agree that moral objections to the research itself and prudent consideration about where it is likely to lead, suggest that they should oppose cloning for biomedical research.
This brings us to the subject of the embryo and what we owe to the embryo. The embryo has always been a puzzle to us. Basically, the embryo is a fertilized egg – a human organism in its germinal stage. It is not just a clump of cells; it is an integrated, self-developing whole, capable of the condiment organic development characteristic of human beings.
Most people believe that it is wrong to allow a cloned embryo to develop into a human being to avoid that wrong, we would have a duty to destroy any such cloned embryo. Advocates of embryonic stem cell research hold that we have a duty to kill it and that it is permissible to start projects that might lead to such mistake and result in such a duty. The reason is because of the kind of moral importance the embryo has. An embryo is not an entity that can be harmed by the loss of its future. This is not stating that we should not destroy the embryo because it is bad for the embryo. We are not saying either that the continued existence o embryos cannot be good for them. After all, an embryo does not have and never has had the capacity to sense, perceive or experience anything.
All are in agreement that the embryo does not yet have, except potentially, the full range of characteristics that differentiates the human species from others; but one does not necessarily have those characteristics in evidence in order to belong to the species. Human beings at any stage of life, do not forfeit their humanity simply because of the lack of evidence of these distinguishing characteristics. There are different points in the life story of any human being – a beginning, a zenith and a decline, but none of these points is in itself the human being. That being is, rather, an organism with a continuous history.
Those who do not subscribe to cloning for biomedical research (position 2) believe that the embryo is in fact “one of us”; a human life in process – an equal member of the species “Homo Sapiens” in the germinal stage of his/her natural development. Those who oppose going forward with cloning for biomedical research maintain their stand that it is not only incoherent but self-contradictory to claim that human embryos deserve “special attention” and yet to endorse research that requires the creation, use and destruction of these organisms especially when done routinely and on a large scale.
If from one perspective, the fact that the embryo seems to amount to little, may invite a weakening of our respect; from another perspective, its seeming insignificance should awaken in us a sense of shared humanity since this was our own condition. Because the embryo seems to amount to so little, our responsibility to respect and protect its life correspondingly increases. Hans Jones maintains that a true humanism would recognize “the inflexible principle that utter helplessness demands utter protection.”
We would be missing something if we stopped with what is owed to the embryo. An embryo may seem insignificant, but that very insignificance tests not only the embryo’s humanity but our own. Even those who are uncertain about the precise moral status of the human embryo, have sound ethical reasons to refrain from using embryos for utilitarian purposes. There are principled reasons why people who accept research on left-over IVF embryos created initially for reproductive purposes should oppose the creation and the use of cloned embryos explicitly for research. There are also powerful reasons to worry about where this research will lead us. All these objections have their ground not only in the embryo’s character but also in our own. Also in concern is not only for the fate of nascent human life, but for the moral well-being of society as a whole.
Cloning for biomedical research and cloning to produce children both begin with the same act of cloning – the production of a human embryo that is genetically identical to its progenitor. Both uses of cloning mark a significant leap in human power and human control over our genetic origins. Both involve deliberate genetic manipulation of nascent human life. If we say “yes” to cloned embryos in laboratories, we are saying “yes” to an ever-expanding genetic mastery of one generation over the next.
The final question to be considered is what we owe to the suffering. Both sides in the debate believe it to be less than human to turn a blind eye to those who suffer and need relief or to stand silent in the face of suffering and premature death. In saying “no” to cloning for biomedical research, we are not closing the door on medical progress. We are just acknowledging the fact that, for very strong moral reasons, progress must take place by means that do not involve the production, use and destruction of cloned embryos and that do not reduce nascent human life to a resource for exploitation.
We are not deaf to the voices of those who desperately want biomedical research to continue. We can feel that desire within ourselves for all of us, for those we love most and who could one day be patients desperate for cure. We know that the relief of suffering, although a great good, is not the greatest good. We all value health and a longer life; however, we also know that life loses its value if we care only for how long we live and not also for how we live.
The scientific enterprise is a moral one, not only because of the goals scientists seek, but also because of the limits they honor. It is precisely the acceptance of limits that stimulates creative advance that forces scientists to conceive of new and acceptable ways of conducting research. Therefore before society takes a step that cannot be undone, it should think seriously of the moral implications of accepting cloning even for research. We must first consider with wisdom and with courage what we owe to the embryo, to our society and to the suffering.
Knowledge and understanding of human biology continue to improve. (Mader, 2004). At first glance, there seems to be no limit to what human beings can do. Even human biology is not beyond man’s ability to manipulate and control. Nevertheless, there are controversial issues. One of the most controversial is the attempt to reproduce an exact replica of a human being through the process of cloning. Human cloning must be banned because people must respect the sanctity of human life.
In the late 1990s, the whole world was astounded by the news that scientists were able to successfully clone a sheep (Pence, 2004). It was supposed to be a scientific breakthrough that would change the world. But after the successful demonstration of the power to clone an animal, there are those who began to fear the inevitable. After the cloning of the sheep, the next logical step is to clone human beings. Before going any further it is important to understand the scientific principles behind cloning.
In its basic form, cloning is the manipulation of a female egg and a donor cell’s nucleus (Cibelli, 2002). It must be pointed out that an egg cell and sperm cell coming from both male and female is a haploid cell (Cibelli, 2002). This means that asexual reproduction is not possible for humans. But cloning can go around this problem by using a complete set of DNA material from one donor. Cloning bypasses the need for a sperm cell to procreate. The DNA can come from any part of the donor’s body. It can come from a single skin cell. The only requirement is that this single cell contains the nucleus and of course the genetic material used for cloning purposes.
Although there is no need for a sperm cell, cloning requires the use of a female’s egg cell. However, only a part of the female’s egg cell is needed because scientists have to remove the genetic material contained within the said egg cell. The complete nucleus of the donor cell is inserted into the empty egg cell. After this procedure, the egg cell looks like a fertilized egg cell because a complete set of genetic material for reproduction is present within this particular egg cell. It is important to highlight this procedure because all the issues that emanate from human cloning can be traced back to this procedure.
There are at least three major issues that can be identified as a counterargument to human cloning. First of all, there is the issue of the need to respect the sanctity of human life. Secondly, there is a major concern regarding the detrimental effects of this procedure because scientists are required to perform numerous trial and error experiments in order to master the science behind cloning. Finally, there are no possible contingent measures to deal with a community full of cloned children without parental care and supervision.
In order to clone human beings, it is imperative to use them as “guinea pigs” similar to the way scientists use laboratory rats in their experiments. In a typical experiment using laboratory animals, scientists can easily terminate the lives of the animals without dealing with the ethical dilemma involved in destroying living organisms. It is an accepted part of human society, to use laboratory animals to test a particular scientific procedure. But the same cannot be said when it comes to human subjects. There is a great deal of controversy surrounding the fact that scientists can handle human beings as if they are mere objects.
Another problematic issue is the outcome of the experiments. The natural way of giving birth to a human being is prone to complications. However, the level of risk involved when it comes to manipulating egg cells and genetic material using Petri dishes, syringes and other inorganic material cannot be calculated. In the event that the baby is deformed the scientists cannot terminate the life of a human being. There is no way to manage this problem.
Finally, there is the possibility of having a community full of children without the love and guidance of a parent. In a world wherein terrorism, heinous crimes and campus shootings fill news headlines, the idea of children growing up without adult supervision is a terrifying thought. Scientists are playing god if they attempt to clone a human being. They will require god-like power to manipulate not only genetic material but the lives of human beings. They must not be given the power to treat human beings as if they are mere laboratory animals.
Human cloning must be banned forever. The scientific community must learn to respect the sanctity of human life. They cannot successfully create a cloned human being without going through a series of experiments that may result in the creation of defective babies. There is no way to justify the artificial creation of deformed and mentally retarded human beings as unwanted byproducts of human cloning experiments. Even if scientists can successfully clone human beings, there is the dreaded scenario concerning the existence of a community populated by cloned children without the love and care of a parent.
References
Cibelli, J. (2002) Principles of cloning. CA: Elsevier Science.
Mader, S. (2004). Human reproductive biology. New York: McGraw-Hill.
Pence, G. (2004) Cloning after Dolly: who’s still afraid? MD: Rowman & Littlefield.