StemCell Therapy

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 wasnt 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 organisms metabolism. Therefore, a cells 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 yeasts 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.

Original post:
Genetic Engineering - an overview | ScienceDirect Topics

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