Genetic Engineering

Genetic engineering is defined as the direct manipulation of an organism's genes including heritable and nonheritable recombinant DNA constructs.

From: Laboratory Animals , 2014

Genetic Engineering

J.S. Robert , F. Baylis , in International Encyclopedia of Public Health, 2008

Introduction

Genetic engineering comprises multiple techniques for the intentional manipulation of genetic material (primarily deoxyribonucleic acid, or DNA) to alter, repair, or enhance form or function. Recombinant DNA technologies, developed in the latter half of the twentieth century, include the chemical splicing (recombination) of different strands of DNA generally using either bacteria (such as Escherichia coli) or bacteriophages (viruses that infect bacteria, such as λ phage), or by direct microinjection. In recent years, these traditional tools have been supplemented by new techniques to design and build – literally, to engineer – novel life forms, generally referred to as synthetic biology.

Genetic engineering, writ large, raises a number of significant ethical issues. In agriculture, for instance, ethicists have highlighted potential human health hazards associated with genetically modified crops and livestock, as well as normative concerns about the treatment of animals and the ecological consequences of genetic engineering. In medicine, there has been significant ethical controversy about the putative distinction between protocols meant to restore function and those meant to enhance function beyond species-typical norms. Additionally, ethicists have attended to the potential human health risks associated with germ-line genetic engineering, as distinct from somatic genetic engineering. Finally, in the context of reproduction, ethicists have argued that genetic engineering raises ethical issues involving the screening and manipulation of embryos to eliminate or introduce various medical and/or cosmetic characteristics.

In relation to public health specifically, genetic engineering raises additional ethical issues concerning not only the potential societal consequences of genetic engineering, but also the wisdom of genetic manipulation of plants, animals, and humans. In pursuit of the goals of health promotion and illness prevention, public health initiatives have traditionally sought to improve sanitation, ensure the availability of clean water, and identify the source of, and develop vaccines for, infectious disease. But with the development of genetic engineering techniques and the sequencing of the genomes of plants and animals (including humans), the scope of possible public health interventions has increased dramatically – but so too have the threats to public health.

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Genetic Engineering

Peter W. Atkinson , David A. O'Brochta , in Encyclopedia of Insects (Second Edition), 2009

Genetic Technologies are More Advanced in Drosophila than in Other Insect Species

One cannot discuss the genetic manipulation of insects without describing the molecular genetic tools that are available in D. melanogaster. Traditionally, a gulf has existed between entomologists who view the harmless vinegar fly as being distant to the problems of insect control and Drosophila geneticists who utilize the many biological attributes of Drosophila to understand the basis of gene action. This gulf will close as comparative genomics reveals similarities and differences in the conservation of many genes and molecular pathways between Drosophila and other insect species. The power of this comparative approach to modern biology will offer insect scientists and traditional entomologists exciting opportunities to bring the power of genetics and molecular biology to the control of insects. The development and application of these tools is what insect scientists seek to achieve in pestiferous and beneficial insects.

Genetic engineering in D. melanogaster is an extremely mature technology. It is founded on several independent phenomena:

1.

The presence of a transposable element, called the P element, which is an efficient genetic transformation vector. This vector has been available and exploited since the early 1980s.

2.

The ability to create and maintain genetic mutants by traditional techniques such as chemical- or radiation-induced mutagenesis or by transposon insertion mutagenesis, and the construction and availability of balancer chromosomes to maintain many of these mutants.

3.

The presence of strains that lack the P element, thus providing recipient strains suitable for P element transformation.

4.

The completion of the Drosophila genome project and the public availability of the data generated.

These planks of achievement are a consequence of the intense and sustained research that has been invested into Drosophila over the course of the last 90 years. The picture in all other insect species is, by comparison, sparse. For example, transposable elements capable of transforming nondrosophilid species have been available only since 1996. Also, traditional mutagenesis approaches have been used to generate mutations in a handful of insect species. Many of these have been lost because of problems arising from the rearing of these species (it should be noted that what attracted T. H. Morgan to Drosophila was the ease with which it could be reared and mated in the laboratory) and, often, because it had been necessary to depend on a handful of dedicated workers to maintain these strains. (In Drosophila, by contrast, there are central repositories for strain maintenance as well as hundreds, if not thousands, of researchers who maintain even the most problematic genetic stocks.) Except for medfly, balancer chromosomes have not been constructed in nondrososphild insects.

Two other factors are important. The interactions, if any, of the transposable elements so far known to transform nondrosophilids with components of the insect genome remain unknown, as do the molecular mechanisms by which these elements move both within and between insect genomes. Second, to date, no insect species other than Drosophila has had its entire genome sequenced. Some mosquito genomes are the target for current and future genomic projects.

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Genetic Engineering

C.A. Batt , in Encyclopedia of Food Microbiology (Second Edition), 2014

Abstract

The advent of genetic engineering has allowed for an unprecedented level of modification of biological systems, including microorganisms. The impact on food microbiology has been significant, including in the area of diagnostics, ingredient production, and the creation of improved starter cultures. There are two major areas of interest: first, the production of ingredients or enzymes for food products or their production using recombinant microbial hosts; and, second, the modification of organisms that are used to produce the foods themselves. In either case, the core set of tools includes a means to propagate the gene to be expressed and a means to introduce that recombinant gene in the host. Issues related to the development of 'foodgrade' organisms are also discussed.

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Genetic Engineering

D.J. Harris , in International Encyclopedia of the Social & Behavioral Sciences, 2001

2 Cloning Genes

All of the manipulations of genetic engineering require muliple copies of the DNA sequence or gene of interest. The original methods of getting multiple copies relied on bacteriophage or plasmid vectors to introduce the foreign DNA into bacteria to produce these copies, as each modified cell produces multiple copies, and the bacterial culture itself increases. This is done by first physically isolating the vector, opening its DNA with a restriction enzyme and binding in DNA from the organism being studied that has also been cleaved with a restriction endonuclease. A new population of bacteria is then infected with the altered vector. Given an appropriate way of selecting the population of bacteria so that it uniformly has the DNA of interest multiplying within, one can isolate a large population of vector molecules with the desired sequence, which is then freed by enzymatic cleavage once again.

Fragments of DNA are identified by physically separating them by electrical charge and molecular weight through gels. The DNA of the vectors and bacteria are generally in the range of one to ten thousand base pairs, and there are a sufficiently small number so that the fragments can be identified with a simple staining technique, usually a compound that binds to DNA and fluoresces under ultraviolet light. The larger quantity of fragments that would be isolated from more complex organisms produces a smear with such dyes, so the base-pairing property of DNA, the obligate pairing of adenine with cytosine and guanine with cytosine that allows for both recognition and synthesis of the linear sequence, is used to identify the same sequence on the gel by labeling a known fragment with an isotope or fluorescent dye. The labeled molecules are called probes. This is also the basis for identifying genetic variation in organisms, either for basic studies or identification of mutations associated with disease.

Isolation of fragments produced by digestion with several enzymes, used both singly and in combination, allows for the construction of a physical, restriction-fragment map. Smaller fragments may be replicated, followed by the chemical analysis of the base sequence within fragments which are then assembled into the final base sequence of the gene. Once the sequence is known, production of useful amounts of a region of DNA may now be done enzymatically in vitro with the polymerase chain reaction (PCR). In this technique, the region between two primers, one from each strand of the final DNA molecule is copied in a logarithmic fashion by a heat-resistant DNA polymerase from a small amount of genomic DNA (it has been done with single cells), using multiple heating and cooling cycles. This technique is also used in diagnostic work (Strachan and Read 1996).

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Engineering Fundamentals of Biotechnology

M. Pyne , ... C.P. Chou , in Comprehensive Biotechnology (Second Edition), 2011

2.08.1 Introduction to Genetic Engineering

With the discovery of DNA as the universal genetic material in 1944 [1] and the elucidation of its molecular structure approximately a decade later [2] , the era of DNA science and technology had officially begun. However, it wasn't until the 1970s that researchers began manipulating DNA with the use of highly specific enzymes, such as restriction endonucleases and DNA ligases. The experiments in molecular biology conducted within Stanford University and the surrounding Bay Area in 1972 represent the earliest examples of recombinant DNA technology and genetic engineering [3, 4]. Specifically, a team of molecular biologists were able to artificially construct a bacterial plasmid DNA molecule by splicing and combining fragments from two naturally occurring plasmids of distinct origin. The resulting recombinant DNA was then introduced into a bacterial Escherichia coli host strain for replication and expression of the resident genes. This famous example represents the first use of recombinant DNA technology to generate a genetically modified organism.

In general, genetic engineering ( Figure 1 ) refers to all the techniques used to artificially modify an organism in order to produce a desired substance (such as an enzyme or a metabolite) that is not naturally produced by the organism, or to enhance a preexisting cellular process. As a first step, the desired DNA segment or gene is isolated from a source organism by extracting and purifying the total cellular DNA. The DNA is then manipulated using numerous laboratory techniques and inserted into a genetic carrier molecule in order to be delivered to the host strain. The means of gene delivery is dependent upon the type of organism involved and can be classified into viral and nonviral methods. Transformation (nonviral, for bacteria and lower eukaryotes), transfection (viral and nonviral, for eukaryotes), transduction (viral, for bacteria), and conjugation (cell-to-cell, for bacteria) are all commonly used methods for gene delivery and DNA transfer. Because no method of gene delivery is capable of transforming every cell within a population, the ability to distinguish recombinant cells from nonrecombinants constitutes a crucial aspect of genetic engineering. This step frequently involves the use of observable phenotypic differences between recombinant and nonrecombinant cells. In rare instances where no selection of recombinants is available, laborious screening techniques are required to locate an extremely small subpopulation of recombinant cells within a substantially larger population of wild-type cells.

Figure 1. Basic genetic engineering process scheme including replication and expression of recombinant DNA according to the central dogma of molecular biology.

Although cells are composed of various biomolecules including carbohydrates, lipids, nucleic acids, and proteins, DNA is the primary manipulation target for genetic engineering. According to the central dogma of molecular biology, DNA serves as a template for replication and gene expression, and therefore harnesses the genetic instructions required for the functioning of all living organisms. Through gene expression, coding segments of DNA are transcribed to form messenger RNAs, which are subsequently translated to form polypeptides or protein chains. Therefore, by manipulating DNA, we can potentially modify the structure, function, or activity of proteins and enzymes, which are the final products of gene expression. This concept forms the basis of many genetic engineering techniques such as recombinant protein production and protein engineering. Furthermore, virtually every cellular process is carried out and regulated by enzymes, including the reactions, pathways, and networks that constitute an organism's metabolism. Therefore, a cell's metabolism can be deliberately altered modifying or even restructuring native metabolic pathways to lead to novel metabolic activities and capabilities, an application known as metabolic engineering. Such metabolic engineering approaches are often realized through DNA manipulation.

The first genetically engineered product approved by the US Food and Drug Administration (FDA) for commercial manufacturing appeared in 1982 when a strain of E. coli was engineered to produce recombinant human insulin [5]. Prior to this milestone, insulin was obtained predominantly from slaughterhouse animals, typically porcine and bovine, or by extraction from human cadavers. Insulin has a relatively simple structure composed of two small polypeptide chains joined through two intermolecular disulfide bonds. Unfortunately, wild-type E. coli is incapable of performing many posttranslational protein modifications, including the disulfide linkages required to form active insulin. In order to overcome this limitation, early forms of synthetic insulin were manufactured by first producing the recombinant polypeptide chains in different strains of bacteria and linking them through a chemical oxidation reaction [5]. However, nearly all current forms of insulin are produced using yeast rather than bacteria due to the yeast's ability to secrete a nearly perfect replica of human insulin without requiring any chemical modifications. Following the success of recombinant human insulin, recombinant forms of other biopharmaceuticals began appearing on the market, such as human growth hormone in 1985 [6] and tissue plasminogen activator in 1987 [7], all of which are produced using the same genetic engineering concepts as applied to the production of recombinant insulin.

As a result of the sheer number of applications and immense potential associated with genetic engineering, exercising bioethics becomes necessary. Concerns pertaining to the unethical and unsafe use of genetic engineering quickly arose with the advent of gene cloning and recombinant DNA technology in the 1970s, predominantly owing to a general lack of understanding and experience regarding the new technology. The ability of scientists to interfere with nature and alter the genetic makeup of living organisms was the focal point of many concerns surrounding genetic engineering. Although it is widely assumed that the potential agricultural, medical, and industrial benefits afforded by genetic engineering greatly outweigh the inherent risks surrounding such a powerful technology, most of the moral and ethical concerns raised during the inception of genetic engineering are still actively expressed today. For this reason, all genetically modified products produced worldwide are subject to government inspection and approval prior to their commercialization. Regardless of the application in question, a great deal of responsibility and care must be exercised when working with genetically engineered organisms to ensure the safe handling, treatment, and disposal of all genetically modified products and organisms.

As the field of biotechnology relies heavily upon the application of genetic engineering, this article introduces both the fundamental and applied concepts with regard to current genetic engineering methods and techniques. Particular emphasis shall be placed upon the genetic modification of bacterial systems, especially those involving the most famous workhorse E. coli on account of its well-known genetics, rapid growth, and ease of manipulation.

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Microbial Biotechnology and Sustainable Agriculture

Sharanaiah Umesha , ... Rajat P. Singh , in Biotechnology for Sustainable Agriculture, 2018

Genetically Engineered Crops: Contribution to Sustainable Agricultural Systems

World is facing serious challenges in the areas of food, health, environment, and energy. To address these challenges, plant genomic is one of the key areas to overcome all these fundamental needs through improving plant genetics and plant–environmental interactions to play essential roles in meeting the chronic demands of global food security.

Genetically Engineered Crops

GE is also called genetic modifications, which deliberates certain characteristics of an organism by manipulating the genetic material of particular plant. GE is also differing from conventional methods of genetic modification in two interesting ways: (1) introduce one or a few well-characterized genes into plant and (2) introduce genes from any species into a plant. Number of crops developed on the basis of genetic manipulation strategies to overcome the food loss and provide food with sufficient nutrition to world. The examples are as follows:

1.

Insect-resistant crops [Bacillus thuringiensis (Bt cotton)]

2.

Herbicide-tolerant crops

3.

Viral-resistant crops

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Transgenes

M.L. Hirsch , R.J. Samulski , in Brenner's Encyclopedia of Genetics (Second Edition), 2013

Introduction

Genetic engineering is the foundation of modern-day scientific research and has been implemented for varied applications, including the creation of multidrug-resistant biological warfare and the development of viral vectors that cure human blindness. The ability to alter an organism's genotype relies on the introduction and persistence of foreign DNA, also known as transgenic DNA. Transgenic DNA can be dichotomized into two types: (1) natural (from another organism) or (2) recombinant (i.e., synthesized cDNA). This article provides a brief review of the design, delivery, persistence, and applications of recombinant transgenic DNA using examples from bacterial, plant, and human genetic engineering technologies.

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Genetic Engineering of Oil Palm

Prathapani Naveen Kumar , ... Devarajan Ramajayam , in Genetic Engineering of Horticultural Crops, 2018

5.8 Genetic Engineering of Oil Palm

Genetic engineering could be the best method to overcome the limitations of conventional breeding and/or to achieve objectives that would be difficult or impossible by traditional means. Attempts are in progress for transforming traits such as disease or pest resistance and quality traits such as oil composition. Genetic engineering could be applied to produce transgenic oil palms with high value-added fatty acids (oleic acid) and novel products to ensure the sustainability of the palm oil industry. Establishment of a reliable transformation and regeneration system is essential for genetic engineering. Particle bombardment was the most successful method of transformation in monocots like oil palm. Agrobacterium-mediated and green fluorescent methods were also tried to improve oil palm through genetic engineering. Upon the development of a reliable transformation system, a number of useful targets are being projected for oil palm improvement. Among these targets are high-oleate and high-stearate oils, and the production of industrial feedstock such as biodegradable plastics. MPOB first initiated genetic engineering to produce high-oleate palms for the industrial feedstock and liquid oil market. The estimated value for high-oleate palms is US$1500/ha/year if the oleic acid content is >65%. More recent targets in genetic manipulation include high-stearate palms such as cocoa butter substitute, nutraceutical oils enriched in palmitoleic acid and lycopene, and biopolymers for industrial applications (Sambanthamurthi et al., 2002). Masani et al. (2014) developed novel transformation protocols based on polyethylene glycol-mediated transfection and DNA microinjection showing that protoplasts are suitable as a target for oil palm genetic engineering. They successfully expressed a reporter gene encoding green fluorescent protein (GFP) allowing the rapid and efficient generation of nonchimeric transgenic calli without the use of standard selectable markers. They observed that 5   mL of DNA (at a concentration of 100   ng/mL) injected into the cytoplasm of protoplasts embedded in an alginate layer was identified as the optimal platform for the transformation of oil palm protoplasts. This resulted in approximately 14% of the injected protoplasts developing into microcalli that continued to express GFP.

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CELLULAR, MOLECULAR, GENOMICS, AND BIOMEDICAL APPROACHES | Growth Hormone Overexpression in Transgenic Fish

R.H. Devlin , in Encyclopedia of Fish Physiology, 2011

Brief History of Genetic Engineering in Fish

Genetic engineering, or transgenesis, involves the introduction of novel DNA into an organism by processes that do not normally occur in nature. Studies in the early 1980s showed that transgenic mice overexpressing growth hormone (GH) displayed a remarkable doubling of body size compared to nontransgenic littermates. These findings powerfully displayed the potential of genetic engineering to modify traits in vertebrates for use in basic science and for applied purposes.

Such growth enhancement was also recognized for its potential to enhance human food production in agriculture, and hence numerous reports soon appeared describing genetic engineering of commercially important livestock, including pigs, sheep, and cattle. Growth responses in these species were much more limited than those seen in mice, perhaps because these other species had long histories of genetic selection for enhanced growth under domestication (see below).

Genetic engineering of fish similarly began in the early 1980s (see also CELLULAR, MOLECULAR, GENOMICS, AND BIOMEDICAL APPROACHES | Transgenesis and Chromosome Manipulation in Fish). Currently, more than 35 species of fish have been genetically engineered with gene constructs designed to alter many traits, including growth, reproduction, disease resistance, and flesh quality. The first report of GH transgenesis in fish was from Dr. Zuoyan Zhu in Wuhan, China in 1986, working with the weather loach Misgurnus anguillicaudatus. In these studies, overexpression of GH genes increased growth in nontransgenic counterparts, and even beyond that seen in the transgenic mouse.

These early experiments kindled a large number of similar studies with fish, primarily using mammalian GH gene constructs, with the objective of generating strains with enhanced growth rate for potential use in aquaculture. Subsequently, the use of gene constructs comprised of fish sequences were developed, which in general functioned more effectively than nonpiscine gene constructs.

GH transgenesis remains a focus of activity for fish genetic engineering, generating strains for basic science investigations of growth physiology, behavior, ecology, and evolution. Some strains are also being actively pursued for application in aquaculture. However, globally, all transgenic fish are currently reared in specialized confinement facilities to prevent fish escape, and none are known to have entered natural environments.

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Genetic Engineering for Strain Improvement in Filamentous Fungi

Sandra Garrigues , ... Ronald P. de Vries , in Encyclopedia of Mycology, 2021

Conclusions and Future Prospects

Genetic engineering of filamentous fungi has been an established approach in biotechnology, affecting processes in many industrial sectors. Non-GMO approaches have the widest applicability as they are not subject to GMO regulations that limit the use of GMO-based approaches in many applications. These regulations differ globally, with e.g., the USA allowing a broader application of GMO strains than the EU. The recent development of novel genome editing technologies, such as CRISPR/Cas9, that enable genetic engineering without the introduction of foreign DNA and without easy trackability has re-activated the discussion on GMO regulations and may open the door for a broader application of these technologies.

The global switch to a sustainable biobased economy provides many opportunities for fungi due to their high ability to convert plant biomass to a wide range of products. Fungal fermentations have the potential to replace many chemical processes that are based on fossil resources, but natural fungal isolates are unlikely to be efficient enough to result in economically sustainable processes. Genetic engineering will be needed to improve the productivity of fungal cell factories, reduce the production of side-products and increase the tolerance of the selected fungi to the process conditions. After the genomics era in fungal research, the coming decade(s) may well become the era of fungal cell factories.

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