Despite the potential benefits associated with gene editing, it has to be strictly regulated and approached with much caution due to its ethical implications, unpredictability, and larger environmental impact.
Al-Balas, Q. A. E., Dajani, R., & Al-Delaimy, W. K. (2020). The ethics of gene editing from an Islamic perspective: A focus on the recent gene editing of the Chinese twins. Science and Engineering Ethics, 26, 1851-1860. Web.
Perspective: For the argument.
Analysis: This article is primarily focused on finding the place for gene editing in the religious minds of believers, in this case, Muslims, if there is one. According to the authors, gene editing technologies can be implemented even though it is unclear as to how they might fit into the religious worldview of hundreds of millions of people. The article will serve as an addition to the existing pile of evidence suggesting the devastating impact CRISPR and related technologies can have on the functionality of human societies as they are today. What makes the source credible is its relatively early publishing date and the feature in an academically-reviewed journal.
Arlidge, J. (2021). Why Jennifer Doudna’s DNA discovery is revolutionising the way we tackle disease. Sunday Times. Web.
Perspective: Against the argument.
Analysis: The source underlines the greatest benefits of gene editing, including elimination of diseases and the creation of more adaptable crops, leading to more regular and reliable harvests. This article will be the primary citation in regards to the many advantages of gene editing. This article is the one of least credible out of the ones present in this annotated bibliography. Yet, it was still selected for the assignment due to it being published in a relatively well-respected newspaper and being written by a senior writer at the publication.
Perspective: Dual perspective (for and against the argument).
Analysis: The article offers readers two opposite perspectives in regards to gene editing supplied by two women, Kierra Box and Tina Barsby, both experts in the field of gene modification and editing as they relate to environmental sustainability. Kierra claims that gene editing remains a largely unexplored subject, which is why there is no efficient statutory framework to oversee its integration into the modern agricultural sector. In opposition to Kierra who supports the implementation of alternative solutions to the problems gene editing is trying to fix, Tina Barsby argues that it is integral not to ignore the pathways scientific innovation provides. The breakthroughs in gene editing allow the humanity to be better equipped to deal with the challenges of food security, biodiversity conservation, and so on. The paper will incorporate passages from Kierra’s statements in order to showcase the downsides of adopting early-day research into practice. Apart from focusing on Kierra’s take on unpredictability, Tina’s point of view will be taken into consideration when constructing the counterargument. It is a highly reliable source published in one of the most prominent agricultural publications rather recently. The viewpoints included in the article are those of industry experts.
Perspective: Dual perspective (for and against the argument).
Analysis: This source is a review article of the book written by one of the most prominent biochemists of the 21st century as well as a revolutionary scientist in the field of gene editing. Dockser Marcus (2017) notes that Jennifer Doudna, one of the authors of “A Crack in Creation: Gene Editing and the Unthinkable Power to Control Evolution,” Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is the future of gene editing aimed at making genetic changes much easier and faster. In regards to the argument the paper is going to make, this article will be used to reference Doudna’s ethical concerns regarding this new technology, which seems to be the primary focus of modern-day gene editing research. This article has been obtained via ProQuest and should be considered a credible source since it was published recently and in a world-renown newspaper.
Hofmann, B. (2018). The gene-editing of super-ego. Medicine, Health Care and Philosophy, 21(3), 295-302. Web.
Perspective: Dual perspective (for and against the argument).
Analysis: The source describes the moral implications of gene editing adoption into modern society in much detail, focusing primarily on the assumptions that are usually associated with bio technological innovations. This article will be utilized throughout the paper to demonstrate that the idea that gene editing is a great option is based upon assumptions, rather than real facts. This source’s credibility stems from it being published in a peer-reviewed academic journal and written by a scientist with years of research experience.
Holm, S. (2019). Let us Assume that gene editing is safe—The role of safety arguments in the gene editing debate. Cambridge Quarterly of Healthcare Ethics, 28(1), 100–111. Web.
Perspective: Against the argument.
Analysis: This article discusses the importance of considering the safety of gene editing as a factor in deciding whether or not such technology should be implemented. The source will be integrated into the paper in the counterargument section to demonstrate how some academics might dismiss the ethical implications of gene editing. The article was written by a prominent researcher and published in a peer-reviewed Cambridge journal in 2019, making it more than reliable enough for this assignment.
Perspective: Dual perspective (for and against the argument).
Analysis: This source discusses the new breakthroughs on the field of gene editing and modification. The author notes that it is now possible to edit DNA quicker than ever, just in a matter of months. This article will be used to demonstrate that the integration of CRISPR and other related technologies is indeed controversial. Firstly, its practical benefits remain unclear. Secondly, there is uncertainty as to how these technologies will even influence DNA since scientists still have no idea about a lot of genes in the body in terms of their functions. This article has been written by Anthony King in 2016 and published in one of Ireland’s most prominent publications, which makes it somewhat credible.
Ormond, K. E., Bombard, Y., Bonham, V. L., Hoffman-Andrews, L., Howard, H., Isasi, R., Musunuru, K., Riggan, K. A., Michie, M., & Allyse, M. (2019). The clinical application of gene editing: ethical and social issues. Personalized Medicine, 16(4), 337–350. Web.
Perspective: Dual perspective (for and against the argument).
Analysis: The source aims at examining the social and ethical implications of gene editing technologies’ integration in depth. The authors make a strong point in regards to the need of regulation and public dialogue when it comes to the subject of gene editing and modification. This article will be used primarily in the background section to the technical terms and introduce some of the social and ethical implications of gene editing. This source is the work of a group of researchers, all experts in the field of genetics and ethics. It was published in a peer-reviewed journal only 2 years ago, making it credible for the purposes of this paper.
Gene therapy is the “insertion or removal of genes which can also be alternated within the cell or tissues of an organism for purposes of treating diseases” (Cross & Burmester). All over the world, “the technique is best known for the correction of defective genes so as to treat diseases; the most common procedural form of gene therapy involves the insertion of the functional gene in order to replace the mutated gene within an organism” (Cross & Burmester).
Over the recent past use of gene therapy has revolutionized the treatment approaches, and research on this subject is justified because of the great potential that this technique offers. Similarly, further research will also shed light on possible risk factors that are associated with this method of treatment. The purpose of this paper is to undertake a cost-benefit analysis of gene therapy as a treatment option.
Summary of the mechanisms of gene therapy
Germ-line gene therapy
Under normal circumstances, germ-line gene therapy procedures involve alternating and replacing all defective genes within the body of an organism (Walesgenepark.co.uk). In this case, functional genes are isolated and inserted into the interior lobes of the reproductive tissues or cells of the relevant organism. Considerably, this therapy operates on the basis that fresh and healthy genes get inoculated into the gametes of an organism in order to reduce the risks of later transfer of the defective genes to the next generation of the organism (Walesgenepark.co.uk).
Moreover, there is the involvement of complete alteration of the genetic makeup of early-stage blastomere so as to enhance changes of the genetic codes which can be passed on from generation to generation. Secondly, germ-line therapy also involves alteration of the gametes before fertilization since, after fertilization, the characteristics are passed on to the offspring’s (Walesgenepark.co.uk).
Somatic gene therapy
Generally, “somatic gene therapy involves the process of alteration of the genetic makeup of somatic cells (i.e., all body cells except sex cells) of an individual” (Rothchild, Laura & Lauren). Contrary to germ-line therapy, there is no transfer of these cellular changes to the next generation of the organism, simply because they are neither sex cells nor gametes which fuse during fertilization (Rothchild, Laura & Lauren). The main focus on the process involves changing the arrangements of the genetic codes of the specific cells using either an in-vitro or ex-vivo DNA delivery system. This technique in today’s life can be employed medically in treatments of a variety of diseases, including; hemophilia, muscular dystrophy, among many others; currently, it is used for cancer treatments (Rothchild et al.).
Discussion of paper and critique of the methods used
Telomerase inhibition strategies
In humans, it’s evident that the telomerase RNA abbreviated (hTR) plays an important role in anticancer therapy. This hTR can be used as an anticancer either individually or in combination with another human telomerase reverse transcriptase (hTERT) (Li, Li, Yao, Geng, Xie, Feng, Zhang, Kong, Xue, Cheng, Zhou & Xiao 4). Current findings indicate that when these two agents combine with the recombinant adenovirus, another nucleotide called small-interfering RNA (siRNA) is formed (Li et al. 4).
Furthermore, it has been established that the levels of telomerase activities together with mRNA, hTR are greatly reduced by the activities of the recombinant adenovirus resulting in inhibition of Xenograft tumor growth (Li et al. 4). This implies that siRNA, which is specifically expressed in recombinant adenoviruses, is the best tool to be used as an anticancer and also in the treatment of oral squamous cell carcinoma (OSCC) (Li et al. 4). The major advantage of this technique is that the anticancer effect on OSCC is virtually accomplished by cellular proliferation in addition to cellular apoptosis (inhibition of tumor angiogenesis) (Li et al. 4).
Monoclonal antibodies in target therapies of breast cancer
Globally, breast cancer is known to be a killer disease that largely affects females; the major causative agent in about 10% of world breast cancer cases is the mutation of the gene, which is inherited from any of the parents (Grammatikakis, Zervoudis & Kassanos 640). Furthermore, the most effective known therapeutic alternative diagnosis or treatment of breast cancer globally is the use of gene therapy. Some of the procedures used in treatment include molecular chemotherapy, antiangiogenic gene therapy, among many other therapies currently being used (Grammatikakis et al. 640).
bacteria-mediated anti-angiogenesis therapy
Most of the recent studies have revealed that there are some bacterial species that are capable of colonizing solid tumors. This inherited characteristic, however, can further be enhanced via genetic engineering, developing a natural anti-tumor activity that always enables the specific bacteria to transfer its therapeutic molecules directly into the target cells (Gardlik, Behuliak, Palffy, Celec & Li 7). There are few completed studies that have completely documented the anti-angiogenesis process, which is basically a bacterial mediated therapy for cancer (Gardlik et al. 7).
There are four recognized approaches that scientists use when utilizing the use of bacteria in cancer treatments; these include anticancer therapeutic-autofiction, DNA vaccination, transkingdom RNA interference, and alternative gene therapy (Gardlik et al. 7). Notable to mention is that the major primary goal of all these approaches is that they all focus on stimulation of angiogenesis suppression.
Argument and future experiments proposed
Based on the evidence of this paper, the discussion focuses on the argument of whether gene therapy is effective when the cost-benefit analysis is undertaken. Personally, I totally agree with the essay topic that gene therapy has more benefits than costs since it has been successful in the treatment of most chronic cancerous diseases. Gene therapy usually works by relying on immunotherapy and the use of vector organisms like viral particles to modify the genome of the host cell that triggers an immune response, which finally destroys the cancer cells present in the body (Cross & Burmester).
By using gene therapy, many cancerous diseases, i.e., lung, prostate, pancreatic cancers, have a higher chance of being completely treated. As such, gene therapy is now emerging to be the most common preferred treatment of choice because of its efficacy over other treatment methods (Cross & Burmester). The gene treatment also allows the use of a single vector or a combination of several vectors aimed at achieving optimal results (Cross & Burmester).
Currently, further research on gene therapy is still ongoing, and various clinical trials have so far been completed, which now has led to the evolution of vaccine productions (Cross & Burmester). The vaccines are promising to be the most effective technique since they only require autologous cells to have them manufactured, although this has many cost implications; this is one of the disadvantages of this technique. The second disadvantage is that very few hospitals globally are capable of having such vaccines manufactured because of the costly technology associated with the production of the vaccine. All these factors ultimately limit the availability of the vaccine as a viable treatment option.
A recent research study provides findings that show evidence of secondary gliomas stem cell, which originates from an astrocytic tumor that contains a genetic mutation that possesses the tumor suppressor gene recognized as p53 (Cross & Burmester). As part of the procedure, it was proposed in the experiments that in order to induce apoptosis of tumor cells, one must incorporate the use of an integrated suicide factor together with the adenovirus-mediated transfer of p53 (Cross & Burmester). Considerably, the main strength of this approach is supported by the fact that p53 mediate apoptosis follows two distinct and separate pathways reducing pathogenicity (Cross & Burmester).
Conclusion
Gene therapy technology has brought a great 21st-century revolution to the modern system of disease treatments, precisely when it comes to cancer treatments. It is remarkable to note that the development of anticancer treatments by modified immunotherapy and gene therapy, among others, has helped to cure many cases of cancers and saved many others from death. However, through gene therapy, many victims of cancer have attained a prolonged lifespan after being subjected to this treatment.
Scientifically, the principle behind gene therapy when it comes to cancer treatment is that a successful cancer treatment involving therapeutic modality always aims at real activation of death pathways within the cancer cells.
Therefore, there is no doubt that for cancerous diseases, the development of genetic engineering and specific cancer vaccines are proving to be the most effective treatment approaches. There are hope and confidence that in the near future, all obstacles encountered during the first generation cancerous and precancerous treatments will be eliminated by the development of second-generation modern therapeutic intervention as far as disease (cancer) treatments is concerned (Cross & Burmester).
Gardlik, R., Behuliak, M., Palffy, R., Celec, P., & Li, C. “Gene therapy for cancer: bacteria- mediated anti-angiogenesis therapy.” Clinigene Current Gene Therapy Weekly (2011): 7.
Grammatikakis, I., Zervoudis, S., & Kassanos, D. “Synopsis of new antiangiogenetic factors, mutation compensation agents, and monoclonal antibodies in target therapies of breast cancer.” Clinigene Current Gene Therapy Weekly (2011): 7.
Li, Y., Li, M., Yao, G., Geng, N., Xie, Y., Feng, Y., Zhang, P., et al. “Telomerase inhibition strategies by siRNAs against either hTR or hTERT in oral squamous cell carcinoma.” Clinigene Current Gene Therapy Weekly (2011): 4.
Rothchild, Allisa., Laura, Martin., & Lauren, Lubrano. “Gene Therapy and the Gametes.” Somatic Gene Therapy. 2010. Web.
Walesgenepark.co.uk. “What is Germline Gene Therapy.” Wales Gene Park. 2011. Web.
Due to the complexity involved in eukaryotic cells, the processes of regulating gene expression may as well be complicated. As a result the process involves many different levels to effectively control the hundreds of cells in a eukaryotic organism. Regulation of gene expression in eukaryotes takes place not only during the process of transcription but also during protein synthesis since the protein produced as the final product performs the function that the expressed gene specifies.
Expression of genes may be regulated during transcription (transcriptional regulation) or after transcription has already occurred (post-transcriptional regulation). Transcriptional regulation involves factors that prevent the coding of a DNA sequence into a messenger RNA or basically the mechanisms control the amount of RNA produced.
The major mechanism used to regulate the transcription process is the control of the binding between the RNA polymerase and the DNA since this bond forms the bridge through which he codes would be sent. Binding of non coding molecules known as repressors to the protein that triggers the binding also controls transcription by altering the structure of the protein to either enhance or prevent the binding process. Certain molecules such as activators and enhancers may also be used in favor of transcription by promoting the bonding of polymerase and DNA to be transcribed. Post transcriptional regulation involves regulating the amount of the mRNA to be translated.
This is done by splicing or capping of the some of the mRNA molecules. Gene regulation in eukaryotes may as well be translational or post translational. Translational regulatory mechanisms work by regulating the amount of mRNA to be processed into proteins. This is done by regulating the transport of these molecules from the nucleus to the cytoplasm from which translation takes place. Factors such as temperature changes have also been confirmed to alter the structures of the binding molecules resulting to antisense binding of the mRNA molecule to chromosomes. After translation has occurred, post translational mechanisms may occur in the cell such as degradation of the protein molecule formed or the proteins may be modified through addition of glucose, acetyl or fatty acid molecules (King, M.W. 2011, p. 1).
Gene expression alterations and cancer
Alterations in gene expression occur through many mechanisms which basically involves changing of the base sequences in the genetic material. One of the major mechanism through which gene expression is altered is addition where a base pair is added to the normal sequence hence changing the specificity of the protein that the code specifies. Addition results to production of a nonsense sequence which does not specify any of the proteins.
Deletion of a base pair(s) is another common mechanism where one or more base pairs are deleted from the normal three base sequences. Similar to addition, deletion results to production of nonsense codes (base sequences which do not specify production of any protein). Another mechanism is substitution which involves replacing a specific base pair with a different one. As a result, the sequence specific for a given protein is changed to another code which in rare cases specifies the same protein or specifies the production of a different protein. Another mechanism is inversion where a base pair turns upside down from its normal position.
This may as well lead to formation of a nonsense sequence. Continued accumulation of these genetic alterations which result to altered expression of genes is what causes cancer development. These alterations are known to cause DNA mutations which in turn disrupt the processes that regulate cell proliferation and cell death. The result is uncontrolled division of cells which accumulate and develop into tumors.
How cancer is formed
The formation of cancer involves several steps. Due to unregulated division of body cells over many successive generations, the cells develop abnormally over and over. With time, they gain new potential to perform certain functions such as release of growth factors and different types of enzymes. As the cells continue growing, they affect their neighboring cells which eventually cause complete or reduced dysfunction of the affected organs. This is usually followed by a critical step where new blood vessels are developed. Through these vessels, nutrient supply to the cancer cells is facilitated as well easy movement of the cancer cells throughout the rest of the body.
The next stage involves development of the solid tumor itself. Although all cancers do not have these steps when developing, these are the general steps that occur in many cases of cancer development. At each of these steps, the cancer cells may progress or may even lessen. However, for cancer to develop completely, mutations must occur and the abnormal cells be alive and continuously dividing as well. Activation of mechanisms to repair DNA can prevent or terminate the development of cancer cells (Ginger, 2008, p. 1).
Genetic changes in cancer cells
The genetic makeup of cancer cells is different from that of normal cells. Some of the genetic changes that occur include presence of one or more copies of chromosomes resulting to alteration in the normal chromosome number. Other genetic changes in cancer cells may even damage the chromosomes. Another very common genetic change in cancer cells is the division of the tumor cells where they divide moving towards the poles opposed to normal cells which divide in opposite directions.
This often results to fusion of three poles into a single daughter cell forming a daughter cell with extra chromosome(s). Continued division of such cells eventually results to production of chromosome sets that are completely different from the normal one. When the cancer cells accumulate during its development, many cells that are genetically different gets mixed up within the tumor and this results to a lower immune response of the patient to chemotherapy and other treatments as well.
These changes are known to affect the behavior of cells since the genes are the controlling factor of cell behavior. Many of the regulatory mechanisms in the body are controlled by genes such as homeostasis and cell division. As we all know, these processes are very vital in the body. Genetic changes in cancer cells may alter the normal pathways that signal, initiate or even regulate these body processes.
Other cancer cells may prevent the cell cycling pathway since all the components that control and regulate this pathway are altered by mutations resulting from cancer cells. Interference of these very vital processes may endanger the body especially when homeostasis is altered because control of sugar and water levels in the body will be interfered with resulting to certain conditions such as diabetes. Cancer cells also prevent apoptosis which involves programmed killing of cells without affecting other cells to regulate their presence in the body for normal functioning (Hoffman, 2010, p. 1).
Gene therapy refers to the “deliberate introduction of genes into an organism” (Lerner & Lerner, 2008).According to Lerner and Lerner (2008), gene therapy helps to correct a genetic defect. In case of a patient suffering from a genetic disease, gene therapy can also be used to ease symptoms (Lerner & Lerner, 2008). In other words, gene therapy entails the use of corrective genetic engineering to correct defective genes.
Biological Basis
Gene therapy is an offshoot of molecular biology. The cell is the basic building block of human life. The cell nucleus is made up of threadlike structures called chromosomes whose basic content is deoxyribonucleic acid (DNA). DNA acts as a carrier of genes. Genetic diseases like sickle-cell anemia are as a result of defective genes. Scientific studies show that individuals with distorted genetic sequence are more likely to be diagnosed with cancer and schizophrenia compared to individuals with proper genetic sequence. In light of this, gene therapy aims to replace dysfunctional or missing protein through the introduction of corrected genes (Lerner & Lerner, 2008).
How Does Gene Therapy Work?
According to the U.S. National Library of Medicine (2014), gene therapy works to correct a defective gene by introducing the corrected genetic materials. Also, gene mutation can lead to loss of protein function and if this happens, gene therapy helps to restore this. Modified viruses (through genetic engineering) act as vectors or vehicles that deliver the gene into the cell.
What does gene therapy accomplish?
Germline gene therapy is among the two widely used types of gene therapy. It entails modifying germ cells genetically. The modified germ cells carry the modification to future generations (Moreland & Rae, 2009). In the other common form of gene therapy (Somatic gene therapy), the modified gene cells are only corrected in the patient and the next generation does not get to inherit them.
Accomplishments
There are a number of examples where gene therapy trials have proved to be successful. A good example is the trial of gene therapy on SCID (severe combined immune deficiency). Follow up studies have revealed normal immune function in patients who underwent the trial. Gene therapy has also been involved in the treatment of various forms of cancer like lung cancer, ovarian cancer, and breast cancer, among others. Trials of gene therapy have also been conducted to treat patients who are genetically predisposed to the development of Alzheimer’s disease, asthma, and breast cancer before the clinical manifestations of these diseases start (Friedmann, 2007).
Social and Ethical Implications
Gene therapy is, for the most part, mainly experimental and as such it poses significant safety concerns. A number of gene therapy applications are aimed at bringing about the “stable modification of the genetic characteristics of an individual” (Giacca, 2010, p. 283). The idea that such modification is ethically sound has led to a heated debate. Gene therapy is largely acceptable as long as it is used to ensure the survival of an individual or to improve his/her health. However, the exploitation of gene therapy for aesthetic, intellectual and/or physical appearance has raised ethical concerns.
The exertion of toxicity to cells by viral vectors is another area of concern. For example the Adenoviral vectors are immunogenic and inflammatory by nature and as such, there is the fear that this could lead to cell transformation. Another issue of concern is the possibility that certain gene delivery procedures could in fact be toxic. For example, during angioplasty where transfer of genes occurs on the arterial wall, the procedure demands that the catheters used should partially block the flow of blood so that the therapeutic nucleic acids can be injected (Giacca, 2010).
Considering that gene therapy does not come cheaply, a number of political commentators have raised concerns that this could give the rich an undue advantage over the poor. In addition, ethicists have expressed concerns about a possible misuses of this technology for other purposes, other than correcting defective genes. For example, there are fears that some scientists might exploit the technology to enhance human intelligence. Since gene therapy entails a modification of the basic human structure, religious leaders are concerned that this amounts to “playing God” and are hence opposed to it.
Risks and benefits
Moreland and Rae (2009) have examined various risks associated with gene therapy application. First, Moreland and Rae (2009) state that in a situation where there is a wrongful insertion of the working gene, this poses a great danger to the patient. Another potential risk could be that the deactivated virus might actually prove to be contagious (Moreland & Rae, 2009). Concerns have also been raised about the possibility of gene therapy causing an inflammation to the patient’s immune system as a response to the new genes inserted. On the other hand, gene therapy is associated with a number of benefits. First, gene therapy has demonstrated huge potential to cure, ameliorate, or prevent diseases with inadequate or no treatment. For someone born with a genetic disease, gene therapy gives him/her a chance to live a normal life.
Personal viewpoint
With gene therapy, there is hope for finding a cure for genetic diseases such as cancer. On the other hand, concerns have been raised about the ethics of using this technology. Nonetheless, somatic cell gene therapy holds a lot of potential in finding a cure to most genetic diseases. For effective realization of the benefits of gene therapy, the government in collaboration with the scientific community and various public associations should develop useful guidelines to govern the use of this technology. People of all walks of life should have access to gene therapy, and not just the rich. Funding of trials in gene therapy should also involve the government and the private sector. In addition, scientists and government regulators need to create awareness on the benefits and possible risk factors of gene therapy application.
Conclusion
Gene therapy involves replacing malfunctioning or missing genes to a patient’s cells with new genes. This is normally accomplished using a virus as a vector or vehicle to transport the new genes to the patient’s cells. Gene therapy is an outgrowth of genetics. The cell is the basic structure of a multicellular organism and each cell has its own definite role. The cell’s nucleus carries pairs of chromosomes which in turn carry genes. In case of any alterations on genes, this is likely to cause genetic diseases like hemophilia or sickle-cell anemia. Gene therapy therefore involves manipulating the gene’s structure through genetic engineering in order to correct such genetic defects.
Since gene therapy entails alteration in the body’s genetic makeup, this has raised a lot of unique ethical concerns. So far, only somatic cell gene therapy has been conducted in different clinical trials. However, there is a concern among ethicists that improvements in the clinical trials on germ line gene therapy could lead to its exploitation to enhance aesthetic, intellectual and physical appearance. Therefore, there is need for players in the fields of government, medicine, religion, biology, and politics to formulate ethical guidelines to govern research in gene therapy.
Reference List
Friedmann, T. (2007). A Decade of Accomplishments: Gene Therapy and the ASGT. Molecular Therapy, 15(9), 1576-1578.
Giacca, M. (2010). Ethical and Social Problems of Gene Therapy. New York: Lerner, K. L., & Lerner, B. W. (2008). Gene therapy: The Gale Encyclopedia of Science, 4th ed. Detroit: Gale.
Moreland, J. P., & Rae, S. B. (2009). Body & Soul: Human Nature & the Crisis in Ethics.Downers Grove, IL: Intervarsity Press.
U.S. National Library of Medicine. (2014). How does gene therapy work? Web.
The FtsZ gene encodes for a protein that plays a role in mitochondrial cell division in bacteria such as E. coli (Birnboim & Doly 2001). The encoded protein, a GTPase, resembles eukaryotic tubulins in both function and structure. The aim of this practical was to isolate the FtsZ gene from a donor plasmid (pProEX) and ligate it into a second recipient plasmid, pBluescript KS II (pBKS II). This process, which involves excising a specific fragment of DNA (foreign genetic material) from a genome and ligating it into a plasmid, is called recombinant gene technology. It was first done in the mid-1970s. Common sources of template DNA for recombinant gene technology include cDNA and genomic DNA.
The practical proceeded in five sessions. The first session involved DNA manipulation, which involved two steps. The aim of the first step, small-scale plasmid isolation (‘Miniprep’), was to isolate a recipient plasmid, pBlue Script KS II (+), from an E. coli culture prepared the previous day. In the second exercise, a double restriction endonuclease digestion of the two plasmids, i.e., the donor (pProEX) and the recipient (pBKSII), was done using BamHI and HindIII. The aims of this step were to excise the gene of interest (Pc-FtsZ) from pProEX and prepare the recipient plasmid, pBKSII, for cloning. The two restriction endonucleases cleaved the DNA at specific sites to generate sticky ends that allowed the foreign fragment (Pc-FtsZ) to be inserted into the recipient plasmid.
Practical two involved the construction of a recombinant vector, pBKS II-ftsZ, using the restriction digests from the first lab session. Its aims were to compare the relative molecular sizes of the restriction digests with standards, confirm whether the restriction digestion was successful, and separate and purify the gene of interest for ligation. It entailed agarose gel electrophoresis to resolve the DNA into separate bands, excision to remove the desired fragments, and ligation of the gene into the pBKSII plasmid. The new pBKSII plasmid contained the Pc-ftsZ gene and could be inserted into E. coli in the next session.
The recombinant plasmids (containing Pc-ftsZ gene) from the above step were used to transform E. coli cells made competent through heat shock. The practical proceeded in two steps: (1) bacterial transformation (cloning) and (2) Southern blotting analysis of the cloned DNA. The objectives were to introduce ligated vector (pBKSII) into E. coli for cloning and analyse the cloned inserts using agarose gel electrophoresis and Southern blotting.
Since not all cells in the last session could take up the ligated vector, identifying transformed cells from the non-transformed ones was important. In the fourth practical session, an analysis of the Southern blot was done using specific probes. The main objective of this step was to confirm the presence of PC-FtsZ in the transformed bacteria. The practical also involved the screening of transformed bacteria using PCR to identify those carrying the pBKS II-Pc-ftsZ construct. The final (fifth) practical involved the screening of the Southern blot (gel) and testing the Taq polymerase colonies to confirm the results obtained in the previous session. Running a gel of the PCR colony screen obtained in the previous practical helped identify specific sequences in the transformed bacteria.
DNA fragments are separated based on size or molecular weight. Once placed in an electric field, fragments of small molecular weight move faster than large molecules (Asubel, Brent, Kingston, Moore, Seidman, Smith & Storuh 1995). This yields a distinctive banding pattern with each band representing a DNA fragment. The bands on agarose gels can be visualised through staining with DNA-staining dyes, such as ethidium bromide, followed by UV illumination (300-nm). In this practical, it was hypothesised that the band sizes will decrease from top to bottom of the gel because smaller fragments migrate further down the gel while large ones only move for short distances.
The blue/white screen is a method used to detect clones bearing a specific recombinant vector (a product of ligation) (Birnboim & Doly 2001). First, a gene of interest (Pc-ftsZ) is isolated from a donor cell and ligated into a vector (pBKS II). The recombinant vector is then used to transform competent recipient cells. The bacterial culture contains a substrate known as X-gal. Positive ‘tranformants’, i.e., bacteria transformed through successful ligation, will form white colonies while those that have not been transformed will remain blue. In this practical, since not all bacterial cells could be made competent through heat shock, it was expected that plating would give rise to both white and blue colonies. In this regard, only the E. coli cells from the white colony were selected for the PCR amplification step in practical four.
The Southern blot technique was used to confirm the presence of Pc-ftsZ gene in the constructed vector. The bound probes were detected using anti-DIG antibodies joined to alkaline phosphatase enzyme. Usually, a purple/brown colour indicates that the probes have hybridised to target DNA. Thus, purple/brown bands were expected where complementary probes were bound to the DNA bands.
Materials and Methods
Mini-Prep Step
This step entailed the extraction of plasmid (pBKS II) from E. coli. First, 1.5 ml of previously prepared bacterial culture was centrifuged at maximum speed for 1 min and the supernatant poured off to generate a dry pellet containing bacterial cells. This was followed by a re-suspension of the pellet in GTE buffer before swirling to generate a homogeneous mixture. A lysis buffer (1% SDS and 0.2 M NaoH) was added to the mixture to lyse the cells and release their contents. The high PH created was lowered using a neutralization buffer (3 M Potassium acetate). The mixture was then centrifuged at maximum speed for 10 minutes. Subsequently, 400 µl of the supernatant was transferred into another tube.
To precipitate the DNA, 1000µL of 100% ethanol was added to the contents of the tube. This was followed by centrifugation at top speed for 10 min to separate genomic DNA (the pellet) from DNA-binding proteins. The pellet was washed in 1ml and again in 500µl of 70% ethanol. This was followed by centrifugation at top speed first for 2 min and then for15 min. The pellet generated was dried using paper towels before being re-suspended in Tris-HCl buffer containing RNase A. The mixture was then incubated at 37°C for 10 min to denature RNA. The pellet was stored on ice for the next step.
Restriction Digestion
This step involved the use of restriction endonucleases to cleave pBKSII and bacterial plasmids at specific sequences. In this step, a mixture containing 3µL BamHI and HindIII, 11µL DH2O, and a buffer was added to two separate tubes holding 10µL and 15µL of pBKS II DNA and pProEX DNA respectively. The ttubes were then incubated for an hour at 37°C to facilitate digestion. Further digestion was stopped through incubation of the tubes at 65°C for 10 min.
Gel Purification
The products of restriction digestion were separated on 1.0% agarose gel that had been stained with µg/ml 0.5ethidium bromide. First, two samples consisting of the pBKS II plasmid digest and Pc-FtsZ insert were placed on the gel containing 5µl loading buffer. The nest step involved running the gel in a tank containing TAE buffer. A MW marker was loaded into the first and last wells on the gel. 20µl of each sample was loaded into different wells. Similarly, undigested samples were loaded into the gel to act as controls. The gel was run at 110V for 50 min to separate the fragments into distinct bands.
The bands corresponding to the plasmid and insert pattern were cut out before being transferred into a micro-centrifuge tube. Its mass was determined before adding QG buffer (three volumes). Subsequent steps involved repeated incubation, dissolution in isopropanol, and centrifugation of the samples at maximum speed to bind the samples to the spin column. The spin column was then washed in EB buffer to elute the DNA. The purified DNA was stored at -20°C for the next step.
Ligation
A mixture of pc-FtsZ, pBKSII, T4 DNA ligase, and a ligation buffer in specified volumes were added into a clean tube. The mixture was then incubated at room temperature for 20 min to allow ligation to occur. It was then stored at -20°C for the next step.
Heat Shock Transformation
Bacterial cells were made competent by first incubating them on ice for 20 min followed by heating to 42°C for 90 seconds. The competent cells were transferred to LB plates containing ampicillin, X-gal, and IPTG to screen for the blue/white colonies.
Southern Blot
In this step, four ‘6 x gel-loading’ samples containing circular pBKSII, dH2O, and DNA fragments (pBKS II and linear pBKS II) were prepared. The gels were labelled with DIG-markers and run at 120 V for 1 hour. Subsequently, the gels were subjected to denaturing conditions (1.5M NaCl and 0.5M NaOH) to degrade the DNA into single strands. After drying them, a piece of nylon membrane was aligned on each gel to transfer the DNA fragments (Southern blot). DIG-labelled Pc-ftsZ probes were used to hybridise cDNA on the membrane. The screening of the Southern blot involved an anti-DIG antibody to detect the DIG-labelled probes. A brown/purple colour indicated the presence of Pc-ftsZ gene on the blot.
Taq PCR Screening
The aim of this procedure was to identify transformed bacteria through PCR amplification of the pBKS II-Pc-ftsZ construct. Cells drawn from the white colony on the LB agar plates were placed in PCR tubes. PCR amplification was then done at different temperatures. The PCR products were separated on agarose gel to determine those bearing the gene of interest. Visualisation was done under UV light.
Results
In the first practical, the white pellet of plasmid DNA obtained was digested using restriction endonucleases to generate pBKS II and pProEX + Pc-FtsZ fragments. To separate these fragments for ligation, gel electrophoresis was done. The gel electrophoresis results are as shown in figure 1 below. The bands indicate fragments of different molecular sizes.
Figure 1: Agarose gel electrophoresis results of restriction products.
In practical three, the ligated DNA was used to transform competent E. coli cells. These cells were grown on LB agar plates containing X-gal and IPTG. Practical four involved the detection of transformed bacterial cells. The cells forming blue colonies in practical three were plated into fresh LB agar plate. The results indicated that out of the five colonies, four formed positive ‘transformants’ while one did not take up the recombinant vector.
Practical five involved agarose gel electrophoresis to screen for the PCR results of the colony obtained above. Four different samples were loaded into the agar wells. These included (from left to right) pBKS II (undigested plasmid), linear pBKS II (restriction digestion products), ‘insert’ (Pc-ftsZ), and ‘ligation’. The sequence in which they were loaded on the gel is as shown in figure 2 below.
Figure 2: The ordering of the PCR products on the agar wells.
After running the gel at 120 V for 1 hour, the bands were blotted onto a nylon paper containing DIG-labelled probes before being viewed under UV light. The banding pattern generated in this practical is as shown in the figure below.
Figure 3: DIG-labelled marker indicating the various fragment sizes.
Discussion
Plasmids confer bacteria with certain essential qualities not encoded by the chromosomal DNA. Their sizes range between 1kbp and 200kbp. The aim of the practical was to isolate the plasmid DNA and use it as a vector for transferring a foreign gene (Pc-ftsZ) into E. coli. The mini-preparation step of isolating the plasmid DNA from E. coli cells involved four main procedures: (1) establishment of a bacterial culture, (2) lysis of E. coli to release cell contents, (3) removal of chromosomal DNA and proteins through precipitation, and (4) plasmid (pBlue Script KS II) DNA isolation. Chromosomal DNA and cellular proteins readily bind to one another to form precipitates (Wilson 1990). In contrast, plasmids, being smaller in size re-nature readily after SDS treatment and thus, come out as the supernatant. Thus, after this procedure, plasmid DNA can be recovered through ethanol or isopropanol treatment to precipitate them.
In this practical, restriction endonucleases, BamHI and HindIII, were used to digest pBlue Script KS II and pProEX plasmids. These enzymes cleave at specific sequences on the DNA molecule to generate sticky ends that facilitate the insertion of a foreign DNA fragment (Wilson 1990). The restriction products are separated on agarose gels, which resolves them based on their molecular sizes. Electrophoresis generates DNA bands of different sizes. In this practical, only those bands that had the right size were isolated and purified.
Figure 1indicates the banding pattern obtained when pBKS II plasmid (digested with HindIII and BamHI and treated with AP) in the first well followed by pProEX plasmid in wells two and three, as indicated by the ethidium bromide staining (red bands). Distinct bands can be seen in the wells containing products cut by the restriction endonucleases and treated with alkaline phosphatase (AP). AP treatment cleaves the phosphodiester linkage joining two sugar molecules that form the DNA backbone. This allows the endonucleases to cut at specific sequences.
In this practical, ligation of the DNA fragment (Pc-ftsZ gene) involved T4 DNA ligase, a bacteriphage that joins free DNA ends by filling the gap between the 3’ end and the 5’ termini with appropriate bases. It requires energy (ATP) and Mg2+ ions, which act as co-factors (Wilson 1990). After constructing the recombinant vector, the next step involved transforming the cells with this DNA. In this practical, the E. coli cells were first selected using ampicillin. Only cells that had the pBKS II plasmid could grow on a medium containing ampicillin. Moreover, LacZ gene, which encodes for β-galactosidase enzyme, allowed for blue/white screening. This enzyme breaks down X-gal (a substrate) into a blue-coloured compound. Thus, cells that contain this gene form blue colonies.
BamHI and HindIII cleave at sites near the lacZ gene. Thus, the insertion of the foreign gene (Pc-ftsZ) into this region will affect the functioning of lacZ gene, such that it will not be able to encode for a functional β-galactosidase protein. As a result, X-gal will not be metabolised into the blue-coloured compound. Thus, such a bacterial cell will form a white colony. In this practical, both white and blue colonies were obtained. To detect positive ‘transformants’ only E. coli cells obtained from the white colonies were selected. Heat shock treatment does not always make all cells competent to take up foreign DNA. This means that some cells will be transformed while others remain unchanged because they lack the ability to take up recombinant DNA molecules. In this practical, the cells were selected based on ampicillin resistance and blue/white screen.
Besides antibiotic and blue/white screening, PCR was used to screen the transformed bacteria, i.e., those that took up the pBKS II- Pc-ftsZ construct. Using two primers (T3 and T7) that are complementary to the vector sequences, the PCR step was done to amplify the foreign DNA. The PCR amplification involved entire bacterial colonies taken directly from the LB agar plate. The DNA from the colonies (template) was mixed with PCR reagents, including dNTPs, primers (T7 and T4), Taq polymerase, and a buffer. PCR involves a DNA polymerase activity that catalyses the replication of a target gene fragment. It proceeds in a chain of reactions that occur in alternating high and low temperatures. The initial temperature is often high to generate single strands from the double stranded DNA. A reduction in temperature allows the primers to bind to the template DNA at specific sequences. Replication of the DNA fragment requires elevated temperatures. In this practical, the PCR products were resolved on agarose gel.
Southern blotting was used to analyse the cloned inserts separated through agarose gel electrophoresis. The technique involved a capillary transfer of the DNA fragments contained in agarose gel to a nylon membrane followed by visualisation and detection of the fragments (Priefer 1994). In the practical, DIG-labelled probes that hybridised to the bands were blotted onto a nylon membrane. Probes only bind to complementary fragments on the nylon membrane. Thus, non-specifically bound probes could be washed off from the membrane using a different elution buffer.
In the practical, because the target fragments and the probes were complementary, washing off unbound probes required elevated temperatures and a strong detergent (SDS) concentration. The detection of the hybridised probes involved the use of anti-DIG antibodies joined to alkaline phosphatase enzyme (AP). AP removes a phosphate group from 5-bromo-4-chloro-3-indolyl-phosphate, which reduces nitroblue tetrazolium chloride into a purple precipitate (Wilson 1990). In this practical, the AS samples turned purple when treated with anti-DIG antibodies indicating the Pc-ftsZ gene hybridised successfully to the probes. The presence of this gene in the PCR products confirmed that the ‘foreign’ gene was inserted successfully into the pBKS II plasmid, which transformed the bacteria.
Practical Questions
Practical One: EX 1A
Ampicillin was included in the growth medium (Luria Broth, LB) to select for antibiotic resistance. The LB medium contained all the nutrients necessary for bacterial growth. However, the inclusion of the antibiotic (ampicillin) ensured that only E. coli cells with plasmids (pBKS II) that confer them the resistance to ampicillin could grow. Thus, ampicillin resistance was used as a selectable marker in this experiment.
‘RNAse’ is included in the ‘miniprep’ step to degrade the RNA molecules in the mixture. Cell lysis releases cell contents, which include bacterial genome (chromosomal DNA and plasmids), RNA, proteins, and other cellular debris. This mixture is treated with ‘RNAse’ enzyme to remove RNA molecules.
Plasmids can be purified from proteins and cellular debris through phenol/chloroform extraction. Ethanol or isopropranol is then added to precipitate the plasmid DNA (Wilson 1990). Affinity chromatography can also be used to bind the plasmid DNA and separate it from the protein/cellular debris mixture. The plasmids are released by altering the pH and salt concentration.
EX1 B
A double (BamHI and EcoRI) digestion of the pBKS II plasmid without the insert (3.0 kb) will yield two fragments because there is a single BamHI site located in the EcoRI cleaving region. It is expected that the fragments will be 1400 bp and 1600 bp in size.
Restriction enzymes have specific recognition sequences where they cleave. BamHI recognises TGG while EcoRI only cleaves the G (guanine) base in GGA. On the other hand, the recognition sequences for Kpnl and Sacl are CGG and GGA respectively.
Practical Two
To visualise the resolved bands, ethidium bromide solution (0.5μg/ml) is added to the gel after electrophoresis. Caution is required when using this approach, as ethidium bromide is carcinogenic. The second approach involves adding the stain to the gel before casting it into slabs. However, this method lowers the migration of the DNA fragments and affects their ‘electrophoretic’ mobility (Brody & Kern 2004).
A ‘submarine gel’ is completely covered by the buffer solution. Thus, loading of samples into the gel as well as electrophoresis occur under submerged conditions. ‘Submarine’ gels can allow an electric current to pass through because the buffer contains ions that carry electrons. Increasing the volume of the buffer prevents the gel from drying, which may affect electrophoretic separation. ‘Submarine gels’ facilitate sample loading and protect the gels from damage.
Samples of undigested pBKSII and pProEX DNA were loaded into the wells on the agarose gel to act as controls. Undigested DNA formed a ‘smear’ while digested samples made distinct bands on the gel.
Ligation involves the insertion of a ‘foreign’ gene fragment into the host cell DNA. For efficient ligation to occur, the fragment and the recipient DNA must have free 3’ and 5’ ends. Blunt ends lack free 3’ and 5’ terminals that are present in sticky ones.
Practical Three: Ex 3A
The cells made competent through heat shock are incubated in LB medium to allow them to take up the plasmid DNA. Therefore, incubation facilitates transformation.
Heat shock involves a drastic change of temperature from 0°C (ice) to 42°C within 90 seconds.
Besides heat shock, bacteria can be made competent through chemical treatment. The E. coli cells are placed in a highly concentrated calcium chloride solution to make them competent.
The plates are inverted before incubation to avoid other bacterial strains that may be present in the water condensing on the lid from contaminating the colonies growing on the LB medium.
Ampicillin is added to the medium to screen for positive ‘transformants’, i.e. those that have the pBKS II- Pc-ftsZ construct.
The addition of EDTA into the DNA strand terminates the synthesis of the probe. This happens because EDTA, a derivative of thymidine, lacks OH at the C-5 end.
Besides Southern Blots, in-situ hybridisation and affinity chromatography can be used to detect the DNA fragments in the transformed cells.
In-situ hybridisation and affinity chromatography are inexpensive and safer compared to Southern blots because they do not involve the construction of probes and primers. However, the strong solutions required to elute the DNA may affect its structure and integrity.
Practical 4A
Besides capillary action, DNA can be transferred through vacuum blotting.
The pre-hybridisation step helps to melt the DNA into single strands.
A membrane can be re-probed if the time and incubation temperature did not allow optimal hybridisation.
Practical 4B
The probe only binds to complimentary bases in the target sequence. Thus, it should be generated from cDNA, not the target DNA. In each PCR cycle, a temperature of 94°C allows ‘denaturation’ of the double stranded DNA to occur, 55°C facilitates the annealing of the primers, and 72°C helps in amplification.
The blue colony is used as a control because it contains untransformed cells.
The Pc-ftsZ gene can also be identified through microarray analysis of the colonies.
Practical Five
Besides the use of radioactively-labelled probes, the DNA sequences can be detected using chemilumniscent dyes.
As expected, a single band is visible in the Southern blot.
Inserting the pBKS II-Pc-ftsZ construct into a suitable vector.
References
Asubel, F Brent, R Kingston, R Moore, D Seidman, J Smith, J & Storuh, L 1995, Current Protocols in Molecular Biology, John Wiley & Sons, New York.
Birnboim, H & Doly, J 2001, ‘A Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA’, Nucleic Acids Research, vol. 7, no. 1, pp. 513-523.
Brody, J & Kern, S 2004, ‘History and Principles of Conductive Media for Standard DNA Electrophoresis’, Anal Biochemistry, vol. 333, no. 1, pp. 1-11.
Priefer, U 1994, ‘Characterization of plasmid DNA by agarose gel electrophoresis’, Advanced molecular genetics, vol. 8, no. 1, pp. 26-37.
Wilson, K 1990, Preparation of genomic DNA from bacteria: Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley Interscience, New York.
Gene expression can be defined as “the synthesis of structural and functional products from information emanating from genes” (Annis et al. 1939). Most often than not, “the products of gene expression are proteins but in non-protein coding, the product may be a structural RNA” (Annis et al. 1939). The establishment of the exact products of the expression of certain genes calls for specialized molecular analytical procedures. One molecular procedure has been reliable in characterizing genetic make ups of even pathogenic bacteria and viruses to aid in definitive diagnosis; Polymerase chain Reaction (PCR). Advances in technology have developed various forms of PCR meant to address various aspects of gene expression. Such forms include qualitative and quantitative PCR (Life Technologies), which are preferably carried out in real time. This experiment made use of the Real-time quantitative PCR to measure gene expression (Fricker et al. 772).
Real time PCR is one of the most powerful and reliable techniques of gene analysis. It is commonly applied in the quantification of mRNA either from endogenous genes or transfected genes of either stable or transient transfection (Fricker et al. 772). RT-qPCR is arguably the most sensitive technique in the analysis of mRNA yet. When using “real-time PCR to study gene expression, scientists often investigate the decreases and increases in the intensity by which certain genes are expressed” (Livak and Schmittgen 404). This is done by measuring the abundance or deficiency of gene-specific transcripts (Fricker et al. 772). This forms the basis of action for this technique of gene analysis. In this experiment, sample tissues were collected and maintained at favorable conditions to ensure the viability of gens. Genes were extracted from the endoplasmic reticulum, which is located intracellular, of the respective tissue cells.
Endoplasmic reticulum’s (ER) main function is to control protein synthesis and secretion. Additionally, it provides the platform on which secreted proteins are translated, folded and assembled (Bailey and O’Hare 2305). These functions are essential for tissues whose functions involve a lot of secretion activities such pancreatic cells and immunoglobulin (Minematsu, Harumi and Naito 542). It is worth noting that the accumulation of unfolded proteins in the endoplasmic reticulum elicits signal transmission from this organelle to the rest of the cytoplasm and nucleus. The amount of accumulated proteins may exceed signals generated to orchestrate increased chaperone expression. This response involves the transmembrane transcription factor ATF6 and two transmembrane protein kinases; IRE1 and PERK (Shen 761). Folding mechanisms are produced by a number of ERs – resident molecules called chaperones and folding enzymes that form the ER chaperones. The latter is involved in the ER-associated degradation (ERAD) mechanism. “The mammalian transcription CHOP is also known as growth arrest, and the DNA damage gene 153 is encoded by a transcription factor that promotes apoptosis in response to uncontrolled ER stress (GADD153)” (Kroeger et al. 7590). Additional to the ER chaperones and the mammalian transcription CHOP, the ER stress induces the transcription factor x box binding protein (XBP-1), which is also called (TREB5). CHOP and XBP1 are mediated by ERSE. ATF6 is produced as a soluble form found in the nucleus; the soluble ATF6 activates transcription of the CHOP as well as ER chaperone genes and XBP1 (Huang et al. 114)
Gene expression analysis
This can be achieved by a number of methods (Northern Blots, Microarrays, Real-Time PCR), all of which essentially measure the expression of genes by quantifying the mRNA levels for the genes of interest. The method used in this practical is quantitative real-time PCR, high sensitivity, reproducibility PCR-based technique, capable of determining the level of expression of genes from a single cell. Two approaches are often used in analysis of data obtained from gene expression experiments using quantitative real-time PCR technique. They include absolute quantification and relative quantification (Livak and Schmittgen 406). Whereas absolute quantification method provides output by relating the PCR signals obtained to those of a standard curve, relative quantification method does the same by relating the PCR signals obtained to those of the targeted gene transcript (Ballester, Cordón and Folch 1).
Objectives
This experiment had an objective to determine the gene expression levels of the genes encoding CHOP/GADD153, BiP and endogenous control 18S rRNA in treated and untreated cells by perform Real-Time PCR using cDNA sample, to examine the expression.
Results
The practical started by making master mixes for CHOP, BiP & 18S rRNA using Sybr Green mix & Gene-specific primers. This was meant to generate a melt curve, which shows the specificity of the experiment method used.
An amplification curve was also generated using the controls. This was meant to establish whether CHOP and BiP are up regulated by Tunicamycin treatment, and what was the level of this up regulation (Ballester, Cordón and Folch 1).
Real-time quantitative PCR was then run using both biological and QR samples of genes, drawing variations between the treated and untreated B104 cells samples.
(Ben-Ari 432).(Ben-Ari 432).
Differences between gene expressions of treated and untreated samples were established by a keen analysis of the data collected, in four well established steps.
(Ben-Ari 432).
(Ben-Ari 432).
Conclusion and Discussion
These findings show a clear up regulation of the biological group treated with Tunicamycin as compared to the untreated group. Perturbation or stress of the endoplasmic reticulum during manipulation induces apoptosis thereby reducing the concentration of genetic materials in the collected samples. On the other hand, treatment by Tunicamycin ensures that endoplasmic reticulum stress is kept at bay hence apoptosis does not occur at an elevated rate (Tahmoorespur et al. 37). Therefore, cells treated with Tunicamycin have higher concentration of genetic material and exhibit higher levels of gene expression (Wyttenbach and Tolkovsky 1213).
These findings perhaps best explain why stress and perturbations in the eendoplasmic reticulum (ER) have repeatedly been cited as the source of neurodegenerative and ischemic diseases and conditions. Endoplasmic reticular stress incites the cells to engage a series of transcriptional response. This response is believed to be a result of protein unfolding and is called unfolded protein response (Wyttenbach and Tolkovsky 1213). The targets of unfolded protein response (UPR) include genetic molecules that characteristically act to minimize the effects of stress and perturbation. This they achieve via the inhibition of the destructive effects of aberrant endoplasmic reticular proteins. However, “severe and prolonged ER stress leads to the activation of an execution program by the cells” (Wyttenbach and Tolkovsky 1213). This stress-induced death in the ER results from the activation of intra-reticular caspase-12. This in turn activates caspase-3, which performs the execution role. Tunicamycin “is an inhibitor of protein glycosylation and acts in that regard to rapidly induce the expression of target genes” (Wyttenbach and Tolkovsky 1213).
Works Cited
Annis, Matthew, Naoufal Zamzami, Weijia Zhu, Linda Penn, Guido Kroemer, Brian Leber and David Andrews. “Endoplasmic reticulum localized Bcl-2 prevents apoptosis when redistribution of cytochrome c is a late event.” Oncogene 20.16 (2001):1939–1952. Print.
Bailey, Daniel, and Peter O’Hare. “TransmembranebZIP transcription factors in ER stress signaling and the unfolded protein response.” Antioxidants & redox signaling 9.12 (2007): 2305-2322. Print.
Ballester, María, Rubén Cordón and Josep Folch. “DAG Expression: High-Throughput Gene Expression Analysis of Real-Time PCR Data Using Standard Curves for Relative Quantification.” PLoS ONE 8.11 (2013):1-5. Print.
Ben-Ari, Elia. “The silence of the genes.” BioScience 49. 6(1999): 432. Print.
Fricker, Michael, Sofia Papadia, Giles Hardingham, Aviva Tolkovsky. “Implication of TAp73 in the p53-independent pathway of Puma induction and Puma-dependent apoptosis in primary cortical neurons.” Journal of Neurochemistry 114. 3 (2010): 772-783. Print.
Huang, Lin, Yaqiu Lin, Suyu Jin, Wei Liu, Yaou Xu and Yucai Zheng. “Alternative Splicing of Testis-Specific Lactate Dehydrogenase C Gene in Mammals and Pigeon.” Animal Biotechnology 23. 2(2012):114-123. Print.
Kroeger, None, None Messah, None Ahern, None Gee, None Joseph, None Matthes, None Yasumura, None Gorbatyuk, None Chiang and None LaVail. “Induction of endoplasmic reticulum stress genes, BiP and chop, in genetic and environmental models of retinal degeneration.” Investigative ophthalmology & visual science 53.12 (2012): 7590-7599. Print.
Livak, Kenneth, and Thomas Schmittgen. “Analysis of relative gene expression data using real-time quantitative PCR.” Methods 25.4 (2001): 402–408. Print.
Minematsu, Takeo, Takashi Harumi and Mitsuru Naito. “Quantitative genotyping by amplifying the polymorphic sequences of Pre-Melanosomal Protein (PMEL17) gene using real-time polymerase chain reaction in chickens.” British Poultry Science [Br Poult Sci] 49.5 (2008): 542-549. Print.
Shen, Shihao. “Widespread establishment and regulatory impact of Alu exons in human genes.” Crossmark 108.7 (2011): 761-767. Print.
Tahmoorespur, Mojtaba, Amir Taheri, Hamid Gholami and Maziar Ansary. “PCR-SSCP Variation of GH and STAT5A Genes and Their Association with Estimated Breeding Values of Growth Traits in Baluchi Sheep.” Animal Biotechnology 22.1 (2011): 37-43. Print.
Wyttenbach, Andreas and Aviva Tolkovsky. “The BH3-only protein Puma is both necessary and sufficient for neuronal apoptosis induced by DNA damage in sympathetic neurons.” Journal of Neurochemistry 96.5 (2006):1213-1226. Print.
Modern society strives for awareness at every step, including giving birth to the best version of children. Around the world raises the number of experiments on reducing the percentage of sick children, as well as modifying genes for appearance and character. The issue causes many controversies since it affects the ethical point of view. On the one hand, oppositionists consider genetic engineering to interfere with the natural balance of the universe, which can lead to global negative consequences. In other words, some people are afraid to bring the situation to absurdity, when everyone can choose the traits. An alternative option is the global improvement of human qualities, which can lead to serious negative consequences. On the other hand, researchers in this field want to help couples that experience health problems or want to change some signs of a child to achieve the desired result. An obvious point is the ability to avoid hereditary diseases increasing the overall level of health. Genetic engineering gains positive effects because it allows the preservation and modification of inappropriate embryos, does not affect parental love’s general attitude, and is not risky.
Modifying genes allows one to get rid of hereditary diseases at the planning stage. At the moment, scientists have determined the genetic roots of many common diseases. The main ones are fatal, incurable diseases such as cancer, Parkinson’s disease, rheumatoid arthritis, Alzheimer’s disease, and others (Kaczmarek et al. 1). As with any research, tests are carried out in a laboratory environment. It means that a woman voluntarily donates her material to conduct an experiment and select the best cells for the birth of healthy children without pathologies. Next, suitable cells are fertilized and placed back into the female body. This process is called in vitro fertilization and helps to avoid genetic diseases. Unsuitable cells were previously discarded, but with the development of genetic engineering, they can serve as material for experiments. If we consider this point of view, essentially unnecessary material allows specialists to make breakthroughs in science and serve as an object of observation to develop genetic diseases under various stimulants.
From an ethical standpoint, skeptics might think that the consumerist perfection mentality will cause parents to love children with unaltered genes less than transformed. Modern society strives for excellence and comfort, and every parent wants the perfect child. This point of view can be refuted by the argument of a long-standing manner of adopting children. Historically, raising a child in a family is the best environment for forming a solid and valuable personality. This procedure is bureaucratic and complex in many countries, but couples still want to help children from orphanages. Many people love adopted children no less because, besides the desire to receive all the best, society craves to help and have offspring, regardless of its original qualities. It is confirmed by the opinion of parents who have their own and adopted children (“Raising Adopted Children”). Kind and responsible people will want to work hard to raise even the most troublesome children and love them as much as they love their own.
Some may think that a person’s desire to achieve the excellence of their children is irresistible and therefore carries risks and dangers. The theory makes sense since the issue has not yet been sufficiently studied. At the current stage of genetic engineering development, there are already positive results of interventions. For example, in 2018, the Chinese twins Lulu and Nana became the first successful experiment with genes protected from HIV (van Beers 1). It can be said that the experiment was carried out “in secret” because of legal restrictions associated with genetic engineering. However, many scientists and stakeholders received an impetus to develop the field when the result announcement took place. The CRISPR method became the basis for further research and attracted the attention and, to some extent, the approval of even skeptics. Thus, freedom of choice is more about education and morality than about the genetic modification of cells.
Genetic engineering is currently a controversial and new topic, with a focus on positive change. People’s opinions are divided into negative and positive since everyone has a different attitude to discoveries and risks due to different mentality and principles. The main counterarguments to cell modification are fears of negative global changes, lack of love for ordinary children compared to genetically modified ones, and the inability to achieve perfection. On the positive side, changing genes leads to the ability to plan for healthy children, free of hereditary diseases, and conduct further experiments for better results. With the right approach and minimal risks, genetic engineering will enable society to save children from incurable diseases and give them a happy life. Legislative restrictions perform a particular problem in this matter; therefore, restrictive laws should be revised for faster and more effective results. It may involve testing volunteers with terminal illnesses. This approach will help scientists to do their job and make the world a better place as soon as possible.
Works Cited
Beers, Britta C. van. “Rewriting the Human Genome, Rewriting Human Rights Law? Human Rights, Human Dignity, and Human Germline Modification in the CRISPR Era.” Journal of Law and the Biosciences, vol. 7, no. 1, 2020, pp. 1–36.
Kaczmarek, James C., et al. “Advances in the Delivery of RNA Therapeutics: From Concept to Clinical Reality.” Genome Medicine, vol. 9, no. 1, 2017, pp. 1–16.
Bacterocera tryoni, a serious pest of diverse fruits and vegetables, belongs to the family of Tephritidae and is native to Australia, particularly the state of Queensland. It is an appropriate model insect that the study used to illustrate genetic linkage and mapping of visible and molecular markers of the white gene. According to Gilchrist et al. (2014), tephritid fruit flies live in colonies that permit sympatric speciation and the maintenance of pure strains. Moreover, B. tryoni flies are easy to manipulate to get desired hybrids because they have a flexible mating pattern that allows male back cross without the interference of crossing over during meiosis. Comparatively, B. tryoni has white recessive marks on its thorax, which are homologous to white eyes of Drosophila melanogaster. The mapping of the white gene on different chromosomes and microsatellite loci suggests the existence of a linkage to the visible white marks.
Gene mapping is essential to create a genetic linkage between the visible white marks and white gene in B. tryoni. In the experiment, male back cross enabled the identification and differentiation of the nature of linkage that exists between the white marks and the white gene. The presence of simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) in the white gene permits RFLP to generate fragments of different lengths. The targeted region of the white gene comprises of 680bp with conserved restriction site (GTAC) and polymorphic restriction site (GTWC) for Rsal. When digested with Rsal, the conserved restriction site in one allele (b) generates two fragments (550bp and 130bp), whereas both conserved and polymorphic restriction sites give three fragments (360bp, 190bp, and 130bp) in another allele (a). RFLP is a molecular marker that effectively differentiates the two alleles of the white gene and enhances the creation of the genetic linkage of white marks. Hence, the purpose of the laboratory report is to describe how amplification of the white gene, restriction fragment lengths, and association analysis lead to the formation of the genetic map that links it to white marks.
Methods
The parental cross was conducted between two stocks of flies to get the first generation progeny (F1). For white marks, male parent flies in one stock were homozygous wild-types with conserved and polymorphic restriction sites for Rsal (wm+/wm+ Ra /Ra), while female flies in another stock were homozygous mutants with a conserved restriction site for Rsal (wm/wm Rb/Rb). Subsequently, the male back cross was done and genotypes and phenotypes of the second generation progeny (G2) were recorded. The ratios of genotypes and phenotypes were determined and compared to expected ones in linked and unlinked genes.
Flies were sampled from parental, the first progeny, and the second progeny and their genomic DNA were isolated. Each demonstration group was assigned 20 samples of genomic DNA constituting of the male parent (MP), female parent (FP), first generation (F1), 16 second generations (G2), and negative control. The polymerase chain reaction (PCR) was done using a thermocycler to amplify the target region of the white gene in the isolated DNA samples of respective flies. The PCR master mix was prepared with the final concentration of 16.6mM [NH]SO4, 0.5mM dNTPs, 67mM Tris-HCl (pH 8), 3mM MgCl2, 0.45% Triton X-100, 0.2mg/ml gelatin, 12.5μM primer mix, and 0.35units/μl Taq polymerase. The amplification was done on a 25μl reaction volume comprising of 18μl, 2μl, 3μl, and 2μl of the master mix, primer mix, genomic DNA, and Taq polymerase, respectively.
The PCR reaction contents were mixed thoroughly and placed in a thermocycler for amplification. The thermocycler was set and the amplification process was allowed to run for about three hours. The PCR conditions were initial denaturation for 3 minutes at 94°C and final extension for 5 minutes at 72°C with cycling times of denaturation for 1 minute at 94°C, annealing for 1 minute at 60°C, and extension for 1 minute at 94°C. After completion of the amplification process, amplicons were removed and stored in a freezer at -20°C to preserve them for the next procedure.
The PCR product volume of 25μl for each sample was divided into two equal portions for genotypic analysis. One portion of the PCR product was digested with RSal for RFLP analysis, whereas the other portion was not digested. PCR products (12.5μl), 1.75 units/µl Rsal, sterile MQ water, and 10X restriction buffer with 100mM MgCl2, 500nM NaCl, 100mM Tris-HCl, and 100mM DTT were used to carry out restriction digest. The mixture of restriction digest (20μl) was prepared by adding 10μl of PCR product, 6μl of MQ water, 2μl of 10X buffer, and 2μl Rsal into a 1.5 ml sterile microfuge tubes for each sample. The digestion mixtures for all samples were incubated in a water bath set at 37°C for 1 hour.
Agarose gel electrophoresis was utilised to analyse DNA fragments obtained from the amplification of the white gene and digestion by Rsal. Gel electrophoresis apparatus constituting of gel tanks, video camera, power pack, UV filter, and UV transilluminator was set up in the laboratory. Moreover, 2% agarose, 1X sodium borate buffer, 25ng/μl pUC19/Hpall standard, and 25ng/μl 1kb ladder were prepared for electrophoresis. The DNA analysis was done by loading 20μl of digested PCR fragments, 12μl of undigested PCR fragments, positive control samples, and genomic standards, as illustrated in Tables 4 and 5 (Supplementary Section). The voltage of electrophoresis was set at 180 and allowed to run until the dye front reaches a considerable distance to allow separation of fragments. Ultimately, the gels were read using UV transilluminator and images were taken for the analysis, as indicated by Figures 1 and 2 (Supplementary Section).
Results
The estimated fragment sizes of undigested and digested amplicons were tabulated in Table 1 and Table 2, correspondingly. Comparison of the estimated and the expected band sizes indicates that the undigested fragments did not deviate significantly from 680bp. The estimated fragment sizes of the undigested amplicons ranged from about 600bp to 750bp.
Table 1. The estimated fragment sizes of undigested amplicons.
Samples
Estimated Fragment Size (bp)
Samples
Estimated Fragment Size (bp)
Male Parent
680
blank
Female Parent
600
blank
F1
1000
blank
G2 S1
G2 S10
680
G2 S2
600
G2 S11
750
G2 S3
600
G2 S12
680
G2 S4
600
G2 S13
700
G2 S5
680
G2 S14
680
G2 S6
680
G2 S15
700
G2 S7
750
G2 S16
680
G2 S8
680
blank
G2 S9
680
No data control
700
blank
blank
Table 2 shows that the estimated fragment sizes of digested amplicons were 550bp, 360bp, 190bp, and 130bp for heterozygous (RaRb), 550bp and 130bp or 360bp, 190bp, and 130bp for homozygous (RaRa or RbRb). These fragments are of the expected sizes based on restriction digest by Rsal.
Table 2. The estimated fragment sizes of digested amplicons.
Samples
Estimated Fragment Sizes (bp)
Samples
Estimated Fragment Sizes (bp)
pUC19/Hpall
1 kb ladder
Male Parent
360, 190, 130
Male control
360, 190, 130
Female Parent
550, 130
Female control
550, 130
F1
550, 360, 190, 130
F1 control
550, 360, 190, 130
G2 S1
550, 360, 190, 130
G2 S10
550, 130
G2 S2
550, 360, 190, 130
G2 S11
550, 360, 190, 130
G2 S3
550, 130
G2 S12
550, 130
G2 S4
550, 130
G2 S13
550, 130
G2 S5
550, 360, 190, 130
G2 S14
550, 360, 190, 130
G2 S6
550, 360, 190, 130
G2 S15
550, 360, 190, 130
G2 S7
550, 130
G2 S16
550, 130
G2 S8
550, 360, 190, 130
No data control
G2 S9
550, 360, 190, 130
pUC19/Hpall
1 kb ladder
blank
The comparative tabulation (Table 3) depicts that phenotypes of white marks link to microsatellites loci of the white gene, Bt1 and Bt7 in chromosome 2, and Bt2 in chromosome 5. While white marks link to homozygous short alleles (SS) in the Bt1 and Bt7 loci, wild-type marks associate with homozygous long alleles (LL) in the same loci. In the Bt2 locus, homozygous long alleles (LL) link to homozygous white RFLP (RaRa), whereas homozygous short alleles (SS) link to homozygous white RFLP (RbRb) and heterozygous alleles (SL) in both Bt1 and Bt2 loci link heterogeneous white RFLP (RaRb).
Table 3. Phenotypes for white marks in the identified microsatellites and chromosomes.
Fly
Sex
wm
Bt1 Chr2
Bt2 Chr5
Bt5 Chr3
Bt7 Chr2
Bt11 Chr6
Bt15 Chr6
Bt17 Chr4
White RFLP
Parent
Male
WT
LL
LL
SS
LL
SS
LL
LL
RaRa
Parent
Female
Wm
SS
SS
LL
SS
LL
SS
SS
RbRb
F1
Male
WT
SL
SL
SL
SL
SL
SL
SL
RaRb
G2-1
F
WM
SS
SL
SL
SS
LL
SS
SL
RaRb
G2-2
F
WM
SS
SL
LL
SS
SL
SL
SS
RaRb
G2-3
F
WM
SS
SS
SL
SS
SL
SL
SL
RbRb
G2-4
F
WM
SS
SS
LL
SS
SL
SS
SS
G2-5
F
WM
SS
SL
SL
SS
SL
SL
SL
RaRb
G2-6
M
WM
SS
SL
SL
SS
SS
SS
SS
RaRb
G2-7
M
WM
SS
SS
SL
SS
SS
SS
SL
RbRb
G2-8
M
WM
SS
SL
SL
SS
SS
SS
SS
RaRb
G2-9
M
WT
SL
SL
SL
SL
SL
SL
SS
RaRb
G2-10
F
WT
SL
SS
LL
SL
SS
SS
SL
RaRa
G2-11
F
WT
SL
SL
SL
SL
SS
SS
SS
RaRb
G2-12
F
WT
SL
SS
SL
SL
SL
SL
SL
RaRa
G2-13
M
WT
SL
SS
SL
SL
SL
SL
SS
RaRa
G2-14
M
WT
SL
SL
SL
SL
SS
SS
SS
G2-15
M
WT
SL
SL
LL
SL
SL
SL
SS
RaRb
G2-16
M
WT
SL
SS
LL
SL
SS
SS
SL
RaRa
Discussion
Genotypic ratios generated from the banding patterns of the digested and undigested amplicons showed that white marks of B. tryoni link to the white gene. Furthermore, comparison of the distribution of phenotypes and genotypes of white marks revealed that they link to Bt1and Bt7 loci of microsatellites in chromosome 2 and Bt5 locus in chromosome 2. Choo et al. (2017) established that the white gene comprises of seven exons that cover different chromosomes and have genetic linkages with white marks. The existence of numerous microsatellites in B. tryoni complicates mapping of the sequence of genes in the two identified chromosomes. In this view, an accurate mapping of the white gene and white marks requires the use of gene knockout and sequencing of target genes (Choo et al., 2017; Costain, Kannu & Bowdin 2018; Gilchrist et al. 2014). Gene knockout aids in linking genotypes to specific phenotypes, whereas sequencing allows identification of chromosome, microsatellite, and gene loci of the white gene.
A critical analysis of the results indicates that agarose gel electrophoresis did not give clear bands, making it difficult to estimate the sizes of fragments resolved. Dowdle et al. (2017) recommend lowering of voltage to 120, accurate loading of samples, and extended time for sufficient resolution of bands in the gel. Modern techniques that could complement RFLP in this study are sequence-tagged sites (STS), fluoresce in situ hybridisation (FISH), and bulk segregant analysis (BSA) (Jagannathan et al., 2017; Pool 2016; Saeed, Wang & Wang 2016). To enhance the understanding of B. tryoni, future research should consider exploring microsatellites and identifying their roles in the inheritance of the white gene, associated genes, and visible markers.
Conclusion
The report reveals that genetic and phenotypic ratios suggest that white marks and the white gene have close linkages based on their patterns of inheritance. Specifically, white marks and the white gene links to Bt1 and Bt7 loci of microsatellites in chromosome 2 and Bt2 locus in chromosome 5.
References
Choo, A, Crisp, P, Saint, R, O’Keefe, LV & Baxter, SW 2017, ‘CRISPR/Cas9‐mediated mutagenesis of the white gene in the tephritid pest Bactrocera tryoni’, Journal of Applied Entomology, vol. 142, no. 1-2, pp. 52-58.
Costain, G, Kannu, P & Bowdin, S 2018, ‘Genome-wide sequencing expands the phenotypic spectrum of EP300 variants’, European Journal of Medical Genetics, vol. 63, no. 3, pp. 125-129.
Dowdle, ME, Imboden, SB, Park, S, Ryder, SP & Sheets, MD 2017, ‘Horizontal gel electrophoresis for enhanced detection of protein-RNA complexes’, Journal of Visualised Experiments, vol. 1, no.125, pp. 1-4.
Gilchrist, AS, Shearman, DC, Frommer, M, Raphael, KA, Deshpande, NP, Wilkins, MR, Sherwin WB, Sved JA 2014, ‘The draft genome of the pest tephritid fruit fly Bactrocera tryoni: resources for the genomic analysis of hybridising species’, BMC Genomics, vol. 15, no. 1, pp. 15(1), 1-16.
Jagannathan, M, Warsinger-Pepe, N, Watase, GJ & Yamashita, YM 2017, ‘Comparative analysis of satellite DNA in the Drosophila melanogaster species complex’, Genes Genomes Genetics, vol. 7, no. 2, pp. 693-704.
Pool, JE 2016, ‘Genetic mapping by bulk segregant analysis in Drosophila: experimental design and simulation-based inference’, Genetics, vol. 204, no. 3, pp. 1295-1306.
Saeed, AF, Wang, R & Wang, S 2016, ‘Microsatellites in pursuit of microbial genome evolution’, Frontiers in Microbiology, vol. 6, no. 1462, pp. 1-15.
Supplementary Information
Table 4: Agarose gel loading schematic for PCR uncut DNA.
Row 1 Lane No.
Sample (20)
Row 2 Lane No.
Sample (20)
1
1 kb ladder
15
1 kb ladder
2
Male Parent
16
blank
3
Female Parent
17
blank
4
F1
18
blank
5
G2 S1
19
G2 S10
6
G2 S2
20
G2 S11
7
G2 S3
21
G2 S12
8
G2 S4
22
G2 S13
9
G2 S5
23
G2 S14
10
G2 S6
24
G2 S15
11
G2 S7
25
G2 S16
12
G2 S8
26
blank
13
G2 S9
27
No data control
14
blank
28
blank
Figure 1. Gel image showing the banding pattern of undigested amplicons.
Table 5: Schematic representation of undigested amplicons loaded on gel.
Row 1 Lane No.
Sample (20)
Row 2 Lane No.
Sample (20)
1
pUC19/Hpall
1
1 kb ladder
2
Male Parent
2
Male control
3
Female Parent
3
Female control
4
F1
4
F1 control
5
G2 S1
5
G2 S10
6
G2 S2
6
G2 S11
7
G2 S3
7
G2 S12
8
G2 S4
8
G2 S13
9
G2 S5
9
G2 S14
10
G2 S6
10
G2 S15
11
G2 S7
11
G2 S16
12
G2 S8
12
No data control
13
G2 S9
13
pUC19/Hpall
14
1 kb ladder
14
blank
Figure 2. Gel image showing the banding pattern of digested amplicons.
Typically defined as the tool for measuring the gene expression ability and, therefore, the tool for fast and efficient diagnosing of cancerous cells (Haferlach et al. 2010), the technology of gene expression profiling is widely used in contemporary medicine. GEP is traditionally performed in thirty-nine steps (Imebaud & Auffray 2005), which include the identification of the experimental design, the collection of genes, identification of samples, array preparation, provision of a targeted synthesis, hybridization, transformation of the key data, acquisition of knowledge and storage of data (Imebaud & Auffray 2005).
A closer look at GEP will show that it basically needs four key stages to be carried out. Particularly, the process requires RNA expression, promoter analysis, protein expression and post-translational modification (Li et al. 2013). It should be borne in mind, though, that GEP has different methods of pattern prognoses (Greaves et al. 2013, p. 261); therefore, some of the GEP process stages may vary depending on the type of prognostic pattern used in the course of the profiling. Therefore, the overall process of GEP includes the following stages: RNA expression (commonly in the form of northern blotting involving the analysis of the mRNA rates, promoter analysis, promoter expression and posttranslational modification analysis (Yoshida et al. 2012). It also should be noted that due to the specific genetic structure of the hyperproliferative tumor, for which aneuploidy is typical, it is rather difficult to “establish correlations between genetic abnormalities and clinical outcomes” (Zhan et al. 2015, p. 1746).
In order to carry out the GEP analysis successfully, one will have to carry out a biopsy with a specimen of the malignant mass obtained. In the case of myeloma, the specimen in question may constitute a sample of the patient’s bone marrow (Lin et al. 2014); in other instances, a biopsy is carried out to retrieve the required specimen and carry out the evaluation of the disease progress.
As a result of the above-mentioned analysis, the information concerning the type of multiple myeloma developed will be made available to researchers. However, in the most successful instances of a GEP analysis, not only the identification of a specific tumor that the oncologist has to deal with but also the classification of myeloma, in general, can be facilitated. Additionally, the above-mentioned analysis allows for locating a more or less identifiable clinical outcome in patients; particularly, the application of GEP in the instances of breast cancer deserves to be mentioned as a tool for defining clinical outcomes and the “previously unknown mediators of the metastatic steps of invasion and dissemination in human breast tumors in vivo” (Patsialou et al., 2012).
Many researchers point out the complexity and the relative costliness of gene expression profiling to be introduced into clinical practice. The problem is complicated by the heterogeneity of multiple myeloma and by the numerous arrays of gene expressions. Different researchers distinguish between five, seven, eight, or even ten subgroups in myeloma molecular classification.
At the same time, it may be recognized that the use of the gene expression profile, which is based on the evaluation of genes, contributing to the processes of proliferation, apoptosis, and cellular differentiation adequately reflects the biological heterogeneity of multiple myeloma. In such a way, it may be used for prognosis or risk stratification. Prognostication, therefore, is carried out based on the outcomes of GEN as the further avenues for addressing the problem are defined. Gene expression profiling may provide the development of new and more veracious approaches to prognosis and improve the efficiency of multiple myeloma diagnostics and, therefore, either merit or demerit the further investigations.
Reference List
Greaves, P, Clear, A, Coutinho, R, Wilson, A, Matthews, J, Owen, A, Shanyinde, T et al. 2013, ‘Expression of FOXP3, CD68, and CD20 at diagnosis in the microenvironment of classical Hodgkin lymphoma is predictive of outcome,’ Journal of Clinical Oncology, vol. 31, no. 2, pp. 256–262.
Haferlach, T, Kohlmann, A, Wieczorek, L, Basso, G., Kronnie, Gd T, Béné, M-C, Vos, J D et al. 2010 ‘Clinical utility of microarray-based gene expression profiling in the diagnosis and subclassification of leukemia: report from the International Microarray Innovations in Leukemia Study Group,’ Journal of Clinical Oncology, vol. 28, n. 18, pp. 2529–2537.
Imebaud, S & Auffray, C 2005, ‘‘The 39 steps’ in gene expression profiling: critical issues and proposed best practices for microarray experiments,’ Drug Discovery Today, vol. 10, no. 17, pp. 1175–1182.
Li, S, Liu, Q, Wang, Y, Gu, Y, Liu, D, Wang, C, Ding, G, et al. 2013, ‘Differential gene expression profiling and biological process analysis in proximal nerve segments after sciatic nerve transection,’ PlosOne, vol. 8, no, 2, pp. 1–10.
Lin, X S, Hu, L, Kirley, S, Correll, M, Quackenbush, J, Wu, C-L, & McDougal, W S 2014, ‘Differentiating progressive from nonprogressive T1 bladder cancer by gene expression profiling: Applying RNA-sequencing analysis on archived specimens,’ Urologic Oncology: Seminars and Original Investigations, vol. 32, no. 3, pp. 327–336.
Patsialou, A, Wang, Y, Lin, J, Whitney, K, Goswami, S, Kenny, P A & Condeelis, J S 2012, ‘Selective gene-expression profiling of migratory tumor cells in vivo predicts clinical outcome in breast cancer patients,’ Breast Cancer Research, vol. 14, no. 5, pp. 139–157.
Yoshida, S, Ishikawa, K, Arima, M, Asato, R, Sassa, Y. & Ishibashi, T 2012, Fibrovascular membranes associated with PDR: development of molecular targets by global gene expression profiling, Web.
Zhan, F, Hardin, J, Kordsmeier, B, Bumm, K, Zheng, M, Tian, E, Sanderson, et al. 2015, ‘Global gene expression profiling of multiple myeloma, monoclonal gammopathy of undetermined significance, and normal bone marrow plasma cells’, The American Society of Hematology, vol. 99, no. 5, pp. 1745–1757, viewed 30 July 2015, via Informaworld database.
The technological advancements of today have led to leaps and bounds in the improvement in human health and ways in which restoration of health can be achieved. Successful gene mapping has led to the identification of the various genetic causes and predisposition of diseases. Scientific knowledge was never this vast before. It can be called a dream come true for physicians and health professionals around the world, who can now inject the right sequences and codons into the genetic system of the host to generate a healthier gene, thereby remedying the disease as well as conferring the patient a lifetime of immunity to that condition.
Background and significance
The role of genetics in the causation and predisposition towards certain diseases has been well known for many years. Yet improper and incomplete knowledge led to gaps in understanding and utilizing this knowledge in practical means. With the mapping of the genes of the human genome, scientists now had a complete map with which to work on. This knowledge was perhaps the beginning of the true gene therapy procedures and the advent of this field. Now gene therapy procedures have created many possibilities. The most common is the placement of a normal functional gene to replace a nonfunctional gene. Other combinations include the swapping of an abnormal gene with a normal one, repairing of the abnormal gene, and the regulation of gene/s.
There are essentially two methods of gene delivery in use. These are categorized as the physical methods and the chemical method, respectively. The chemical methods employ various chemicals for the introduction of DNA into the body in order to transfer substances from one place to another. These include the DEAE Dextran, which is a polycationic derivative of dextran, calcium phosphate, cationic lipids or lipofection, polymers, dendrimers, and targeting proteins and peptides.
The initial method of gene delivery that is still widely used today is the viral method. Viruses are used as vectors due to their inherent properties to combine and reconfigure the human genome. Instead of infecting, these viruses are prepared and inserted to carry out a particular task, as mentioned above.
The researchers are, however, still looking for novel ways with which to introduce these genes into the body due to the health and safety concerns as well as the biological and ethical issues involved. The use of polymers, peptides, etc., are all in line with the use of nonvirus vectors in the delivery of these genes.
For a successful gene vehicle, it must possess four qualities. It should have the ability to tightly compact and protect the DNA injected, the vector should be recognized by the target-specific cell-surface receptors, and it should be able to disrupt the endosomal membrane, and finally, it should be able to deliver the DNA unharmed to the nucleus. Any substance that can accomplish these four goals is an ideal substance for gene delivery.
Of the viral technique, the four viruses currently in use for the gene therapy include the retroviruses, the adenoviruses, the adeno-associated viruses, and the Herpes Simplex viruses. Some other viruses now being used also include Lentiviruses, adeno associated viruses, and vaccinia viruses.
The ultrasound method of gene delivery is another method with which transfers have been successfully achieved. This method is one of the successful physical methods of gene delivery, which have shown good results and a 10 to 20 fold increase in the permeation of the genetic material. Although it is dependant on certain factors such as frequency and the strength of the ultrasound and the amount of plasmid used with the DNA, the results are almost always beneficial. It is a good noninvasive method used in the transfer of genetic material and therefore is an area of immense interest.
Ultrasound is an example of the physical methods of introduction of genetic material into the body. Many other physical, genetic methods exist, which include microinjection, electroporation, gene gun, and naked DNA.
Current state-of-the-Art
The gene delivery method is primarily chosen on many factors regarding the location and the site of DNA injection. The first and foremost concern is the type of cell environment where these DNA molecules will be injected into. In this regard, the main concern lies in the selective uptake of the molecules and not letting absorption take place in other cells. This is an area that has limited the full potential use of gene therapy. In this situation, some of the more popular methods are the use of peptides, viral vectors, retroviruses, and cationic lipids.
The second problem is the correct identification of the DNA uptake capability of the tissues with which one is working. This is because some tissues take up the DNA with more difficulty than others. And therefore, prior knowledge about the tissue nature should be known before any therapy is carried out. Delivery efficiency must be calculated, which is usually the best with the viral vectors used for DNA transfer.
Next, the type of gene delivery is important, whether it is stable or transient, and what kind of nucleic acid is to be introduced into the body. The selection of the right method can help prevent the death of many cells during the procedure; for example, the electrophoresis and the gene gun delivery systems are extremely harmful to the cells and cause cell death in a majority of these. The suspension cells are more difficult to gene transfer than the adherent cells, and other considerations such as expertise, time, and cost of the procedure also govern the outcomes of the gene therapy success.
The new possibility of the introduction of polymers in the gene delivery methods is under investigation. This is a deviation from the road in which only live vectors or viruses have been considered to be probable vectors into the body. The polymers are the first of the kind to be introduced into the body, which is synthetic and nonliving. This is specifically being done so to reduce concerns about injecting viruses into the body. For this purpose, not every polymer can be used, and those that could be must be further modified to enhance the properties and outcomes. The current blue-eyed polymers for the investigators include the beta-amino esters, which carry the potential of becoming gene carriers. This is because they behave in a somewhat similar manner as do viruses when they enter the bloodstream. Again, these researchers believe that by changing the morphology and sequence arrangement of the molecules, they can improve the outcomes of the performance levels. In vivo studies in mice have been successful so far, and therefore, they hold promise in their use in human models.
This is because these polymers are able to retain themselves with the DNA, enter the cells successfully, and instead of being broken down by any enzymes, enter the nucleus and exert their genetic effects and results.
Some of the other methods of gene delivery currently in use include the direct injection of the DNA into the target cells and thereby achieving effects. Others can include the use of liposomes within aqueous mediums, which carry the therapeutic DNA, and binding the DNA molecule to specific molecules so as to attach itself through specific target receptors.
Another possibility of using nonviral vectors is the use of peptide agents in the delivery of the gene sequences. This area of research again was introduced to gain independence from the viral methods of gene delivery and has already shown promise in this regard. Peptides have been able to show almost the same qualities of gene deliverance as in the cases of the virus, without the need to use the virus, making it an active area of research. Lysine and arginine-based peptides are able to make themselves more compact, which can be stable for a long time within the serum. Similarly, targeting and attachments can be carried out with precision, which was not possible with many of the other new agents developed. The reduced cytotoxicity and immunogenicity and increased biodegradability make these substances ideal for gene therapy procedures.
With the advent of gene therapy, many new procedures have been introduced. Each one has shown positive contribution and results. For example, Pathway IV gene delivery mechanism is a very good system introduced for patients who have any muscular problems such as muscular dystrophy. It has become a popular method due to its ease of administration and the long effect on the single dose. Intravenous plasmid DNA is injected into the venous system into the area of muscle to be treated. This plasmid gains residence in the targeted tissues, whereby it causes changes in the muscle by producing proteins locally or systemically to cause repair. The complexity may vary to the level of including antigenic and immune responses. While this is a good method of delivery, the only problem is the somewhat inability to specifically target the diseased tissue only.
A somewhat recent method of overcoming this problem was introduced to increase the vascular pressure so as to aid the delivery of the substance into the specific tissue. This has two benefits. Firstly the nucleic acids, which in normal circumstances may not be absorbed by the cells, are done so quickly, and secondly, to the target tissue. This can be easily done with the help of a tourniquet and diseases such as muscular dystrophy, anemia, cancer, etc. The applications are variable, and either local or systemic, and the outcomes are positive.
This method was an example of gene transfer by needle injection of the naked DNA(5). This technique is primarily carried out in tissues such as the lungs, liver, muscle, and skin. However, they show low gene expression. Again the issue here is the nonspecific mechanism of binding to the tissues and target cells. While this therapy is very useful in some tissues, it is not feasible due to the hydrophilic nature of the DNA, which prevents its entry into the cells. A technique to increase this permeability is the introduction of various other substances that enhance the DNA’s permeability. These include substances like transferrin, water-immiscible solvents, non-ionic polymers, surfactants, hypotonic solutions, and nuclease inhibitors.
There are many challenges that are causing problems in the progression of gene therapy at the moment. All of these are concerned with the complexity of the human body and the mechanism and the way with which it responds to external stimuli. The first challenge that scientists are facing is the short-term results that are achieved in the gene therapy introduction. The genetic results anticipated are hard to achieve in the dynamic environments of the dividing cells. Therefore, the results are achievable for a limited time frame only. In order to truly succeed in gene therapy, it is important that the genes introduced become fully integrated into the cell’s genome and act as if it was the body’s own.
The second issue is the immune response the body may elicit towards the foreign material. Third, the viral vectors have the potential to achieve their potential for causing disease within the patient and may cause other body defense systems to get activated. Lastly, while single gene dependant diseases have shown promise in the treatment and cure of the condition, not the same could be said for those conditions that are dependant on the multiple genetic variations or discrepancies. This complicates the treating of such diseases considerably and is an area of intense research for scientists.
Improvements
A novel possibility of creating a 47 chromosome cell with an extra chromosome to already present 46 chromosomes is not a very new idea and has been in processing for some time now. Scientists claim that it will take an autonomous chromosome not able to elicit anybody’s response and will contain the genetic information as a vector. There is, however, much research going on in this area.
Of the current technologies, the use of transposons is becoming the next method of delivery of genetic material. Also nicknamed the jumping genes, the transposons are superior to either viruses or a plasmid, which are currently underuse. These have the advantage of not being infectious as in the case of viruses and getting to the target tissue, which is not as efficient in the case of plasmids. These transposons can be used to carry one desired DNA sequence to another one and thereby enzymatically activate or deactivate it. A somewhat newer area of research, it has to undergo the same scrutiny procedures to make it safe to use in man successfully.
The current advancement in genetic transfer therapy is being investigated by the Innovation Corporation. The use of genetic vaccines is in studies and researches. Although not exactly a gene therapy, this method is essentially a protein recombination technique. This method does not use gene transfer and therefore prevents the risk of getting any mutations or reactions from the therapy. This therapy has shown a longer duration of action when compared to standard gene therapy procedures and can be effective for as long as six months. These vaccines have the advantage of using the genetic component of the disease only, which causes the production of proteins. These proteins then cause the activation of the body’s immune response that acts to kill the diseased or cancerous cells.
Gene delivery methods have been of immense interest in the fields of cancer chemotherapy. Here poses a great challenge for the researchers; for a while, they know which genes need to be altered to cause the death of the cancer cells, the methods of delivering these DNA sequences to the cells remain a feat to be achieved. In solid tumors, this becomes an exceptionally difficult issue. The extracellular barriers include the nature of the solid tumor, wherein diffusion becomes dependant on the properties and nature of the molecules, the kind of virus used for the transfer, and the composition and structure of the tumor. The higher than normal intracellular pressure of the cells of the tumor mass increases the difficulty of penetration into the tissues. In summary, these barriers are the interstitial penetration and transportation, and cellular targeting.
In this situation where the intracellular pressure is high, the use of intratumoral injection has been considered a good alternative. Injection directly into the tumor site with high pressure aids and increases the chances of the delivery of the molecules into the cells and thereby achieving effect. The only problem in this method is the leaking of the viruses injected into the systemic circulation, which can give rise to systemic symptoms. For this purpose, attempts are being made to inject the viruses in a substance that is highly viscous, so as not to allow the diffusion of the viruses into the circulation. This has been successfully achieved through the alginate solutions, which have an extremely high viscosity, and decreased rates of diffusion. Th se findings have given scientists much to hope for, in an attempt to treat cancer patients with permanent outcomes. However, the technical limitations are still holding outcomes that are desired. (10)
Conclusions
Gene therapy holds much promise for the future treatment of many conditions that are genetic in nature, as well as cancer patients. The present research has been able to identify the technical difficulties that are associated with this method. However, given the growing understanding of the process, it is now ascertained that the future will be an era for the genetic therapies, and cures.