General Applications of Genetic Engineering

By: Bijay Dhungel , MSc

Introduction

Gene cloning has had a massive impact in basic biological research as well as the everyday life of the general public. In this article we will discuss some basic biological research, as well as the areas of therapeutic cloning and gene therapy. We’ll also delve into the topic of Genetic engineering to produce recombinant human proteins, and Genetically modified organisms and the fascinating world of Transgenics.

Basic Biological Research:

basic-biological-research-future-human-evolutionFor any application, scientists require thousands or even millions of copies of the same gene in a test tube. Once this has been achieved, it can be used for various purposes.

When a gene is cloned in a vector, it becomes possible to sequence the gene—the process of finding out the precise order of bases that appear in the gene. This is possible because DNA is made up of four bases: adenine, guanine, thymine and uracil. The sequence of these bases in the DNAdirects the process of transcription–the production of mRNA that in turn dictates the sequence of amino acids in the protein. Thus, gene sequencing is an important part of basic biological research. There are public databases where scientists deposit the sequence of genes once they are sequenced both for public and scientific use. After sequencing, it is possible to know which protein the gene makes.The genetic code is applied to determine the precise order of amino acids in the protein. The sequence of the protein is then compared with all the known proteins (over 50,000) in databases like Swissprot Uniprot and others, which are databases used to house biological information about known proteins for public and research use. Comparisons can be easily done using computers and software that are available in nearly every lab. If the unknown protein is similar to one in the database, it is possible to guess the function of the gene. Even if there are no identical proteins, there are many other ways to guess the function of the gene once it is cloned (1,2,3).

An example of this application is the identification of the gene responsible for cystic fibrosis. This gene was identified in 1989 when it was cloned and the protein it coded was found to be responsible for calcium transport in lung cells. If this protein is defective, calcium transport is deficient and mucus accumulates in the lungs leading to cystic fibrosis. Once the gene responsible for disease is known, it is possible to begin experiments aimed at discovering a cure. There are many other diseases which are caused by a defect in a single gene, and genetic engineering has made it possible to identify them(4).

Therapeutic cloning:

Therapeutic cloning is a process in which nuclear material is transferred (nucleus) from an individual into an enucleated egg (an egg whose nucleus is removed) to derive embryonic cells with the same genome as the nuclear donor. Once the embryonic cells are formed, they are grown to stem cells. These stem cells can now be used to grow replacement organs like heart, lungs and so on by a process called organogenesis. And since the genetic material of the organ is derived from the donor, the problem of immune rejection is circumvented. In fact, laboratory studies show that this method holds immense promise in the treatment of diseases like Parkinson’s disease, Duchenne Nuscular Dystrophy and Diabetes Mellitus. Moreover, combined with gene therapy, therapeutic cloning could be used in treatment of many other genetic diseases. The basic steps for therapeutic cloning arestraightforward: first,DNA is extracted from a sick person. It is then inserted into an enucleated donor egg, causing the egg to divide, similar to a fertilized egg. This forms an embryo, the place where stem cells are collected. Finally these stem cells may be used or grown to any kind of tissue or organ for treatment of the patient. However, many technical and non-technical (legal, ethical, political and economic) concerns that must be addressed before therapeutic cloning can live up to its expectation (5,6,7).

Gene therapy:

When a person falls ill, it is often due to an invading microbe which damages or destroys cells and organs in the body. Diseases like cholera, AIDS, diphtheria, measles, and the common cold are a few examples of this type of illness. These types of diseases are called infectious diseases. When a person suffers from a infectious disease, the physician prescribes a drug which fights the microbe and often removes the microbe, thus curing the sick person. However, most of the diseases people suffer from are not of this type. The diseases that are caused by alterations in genes are termed genetic diseases. These diseases may arise due to one or more mutations in a normal gene that eventually lead to a nonfunctional protein or no production expression at all. Thus, the biochemical pathways that the protein is involved in are disturbed, and the disturbance manifests itself as genetic disorder. There are several thousands of genetic diseases that cause serious threats worldwide, such as cancer and cystic fibrosis.

Gene therapy is a simple approach to cure genetic diseases. If a disease is caused by a faulty gene, it must be possible to cure the disease by repairing the gene. Many approaches can be used to achieve this goal. The most common approaches are to add a normal version of the gene or cells that express the normal version of the gene to the patient’s body.. Although gene therapy is simple and straightforward in theory, there are numerous technical problems that can arise when trying to repair the faulty gene. Identifying the faulty gene, delivery of the normal gene, controlling gene expression, and duration of gene action are among the main technical problems that still need to be addressed for gene therapy to become the forerunner of treatment of genetic disorders (8).

Genetic engineering to produce recombinant human proteins

As we know, proteins are the real “workers” in the cell. Proteins are required for structural integrity, function, regulation and survival of tissues and organs. Without protein there can be no cell, and without cell there can be no life. Proteins can act as antibodies, which recognize and destroy invading pathogens like bacteria and viruses, thus helping to protect the body. They are enzymes that carry out all the biochemical reactions happening inside the body. They are also indispensable for maintenance and flow of the genetic code. They act as messengers (e.g. hormones) to transmit signals between different cells, tissues and organs. Proteins that provide structural integrity to organs help us to move. Proteins also transport molecules throughout the body per the body’s needs.

Given the immense importance and versatility in function of proteins it is easy to imagine that they are widely used in research, medicine, and industry. But the extraction of proteins from the natural source is an extremely delicate, difficult and expensive process. Genetic engineering provides an alternative to this problem by making it possible to transfer the gene that codes for a particular protein and express it in either a prokaryotic or eukaryotic host. This is done by putting the gene of interest in a vector with promoters, enhancers and other sequences required for the expression of the gene in host cells. The vector is then introduced into hosts like bacteria, yeast or mammalian cells that produce a large amount of protein (9). The applications of recombinant proteins is vast. Recombinant proteins can be used in protein replacement therapy if aberration in a protein is identified as the cause of a genetic disease, and the recombinant protein can be used for its treatment. Insulin was the first protein  produced by genetic engineering, and the recombinant insulin can used for the treatment of diabetes mellitus (10,11,12). Dwarfism is also a genetic disorder–it is caused by the underproduction of human growth hormone in the body. The synthetic human growth hormone can make up for the body’s deficiency of the hormone (13). Similarly, bacteria-derived interferon was shown to reduce symptoms of hepatitis, decrease the spread of herpes zoster, and shrink certain tumors (14). Many other recombinant proteins have been expressed and patented by companies. Some of the examples include: Erythropoietin (a protein that controls the formation of red blood cells) patented by Amgen (15,16), Tissue plaminogen activator (protein responsible for breakdown of blot clot) patented by Genentech (17,18), and Aldurazyme (enzyme that degenerates glycosaminoglycans in lysisimes) by BioMarin Pharmaceutical and Genzyme (19,20).

Genetically modified organisms/ The Fascinating World of Transgenics

Genetically modified organisms (GMOs) are organisms whose genetic makeup has been altered in such a way that it would not occur naturally (through mating and/or natural recombination). Genetic engineering provides an option to transfer a selected gene or genes from one organism to another. The gene that is inserted into a foreign organism is called a “transgene” and the organism made during the genetic modification process is called “transgenic.”There are several approaches that can be used to make a transgenic organism. In the first, the desired transgene is identified and cloned. Then it needs to be inserted into the target organism. To make transgenic animals, the desired gene is micro-injected into the reproductive cell of the organism, the cell is grown in vitro (in the lab) to embryonic stage, and then inserted into a centerpiece female where it is nurtured and a modified organism is born. Another approach is to use retroviruses that have been crippled so that they cannot cause infection, but carry the transgene and inject it directly into the host cell where the transgene incorporates in the host genome. The third approach is to use totipotent stem cells, which are cells that can grow into any cell (like liver cells, neuronal cells and so on). In this approach, totipotent stem cells are isolated from the host’s embryo, injected with the desired transgene, and reinserted into the host embryo. This embryo is then grown to a genetically altered adult (21). Unlike animals, all of the cells in plants retain the capacity to develop into a new plant, thus the transgene maybe inserted by any of the transfer methods into a single cell. Tissue culturing is then done to propagate the cell such that multiple cells express the transgene, and the development of transgenic plant is possible. Once the plant is made, nature increases the plant number through normal seed production and propagation (22).

Transgenics has a widespread application. Given the population explosion over the last 100 years, coupled with the decreased availability of land for farming has led to a high demand for food. At present, crop yields are barely keeping up with demand. Genetic engineering of plants has the potential to overcome this problem through the sustainable and safe production of food with increased nutritive value, better flavor, prolonged freshness and even disease fighting abilities. Moreover, it is possible to make crops with higher yields which are naturally resistant to insects and pests. This particular ability of genetic engineering helps to protect the soil by decreasing or entirely removing the need for insecticides, pesticides, herbicides and fertilizers. There are numerous examples of GM crops with better qualities. Fruits have been produced which ripen on the vine for better taste yet have a longer self life. Today, genetically engineered bacteria is used to produce an enzyme called chymosin that is used to produce most  cheese products. Increasing the level of iron in rice is a potential remedy to combat the problem of iron deficiency that affects over two billion people worldwide.. A company called Monsanto found a way to increase the starch content of a potato, which has the advantage of reduced oil absorption during frying, thus lowering the cost of French fries, chips, and so on, while  at the same time reducing the fat content. In addition, since the production of is, Scientists are also trying to produce edible vaccines in tomatoes and potatoes to help with the issue of a tiresome, long and expensive process to produce medicines and vaccines (23).

Similarly, transgenic animals have a widespread application in agriculture, medicine, and industry research.. Selective breeding to produce animals exhibiting desired traits like increased milk production and higher growth rates has been practiced. However, selective breeding is difficult and time consuming. Genetic engineering solves these problems by using tools that make it possible to develop the desired traits in animals in a relatively short time and with more precision. For example,transgenic cows that produce more milk or milk with less cholesterol or less lactose; Sheep that can grow more wool;pigs and cattle that produce more meat are already available (24). Moreover, scientists are working on making disease resistant animals like cows resistant to intramammary Stephylococcus aureus (25). Transgenic pigs may be used to produce transplant organs like hearts, livers, and kidneys. This process is called xenotransplantation. However, there is still a problem of organ rejection that needs to be addressed (26). Transgenic animals may also be used as “bioreactors” to produce important and valuable human proteins, enzymes, hormones and growth factors. This method is a cost effective alternative to cell culture method discussed above (27). Various research has been done to explore the possibility of production of growth hormones, anti-clotting factors, and insulin from the milk of transgenic animals. In particular, scientists are working on producing milk for the treatment of diseases like phenylketonuria, cystic fibrosis, and hereditary emphysema (28). Perhaps the best example of industrial application of transgenic animals is the production of “Biosteel” from transgenic goats. Nexia, a Canadian biotech company has produced trangenic goats that produce spider silk protein that is stronger and more flexible than steel. This silk is so strong that it could be used to make bullet proof vests, and may also have applications in the aerospace industry. Owing to its compatibility with the human body, this silk may further be used to help tissue repair. Transgenic animals can also be used in research to answer fundamental questions in biomedical sciences like gene expression and regulation, developmental biology, immunology, and cancer research, and at the same time they can be used as models for human genetic diseases like muscular dystrophy, and sickle cell anemia among many others (28,29).

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