The Black Unknown 2.0

Genetic engineering, recombinant DNA technology, genetic modification/manipulation (GM) and gene splicing are terms that are applied to the manipulation of genes, generally implying that the process is outside the organism's natural reproductive process. It involves the isolation, manipulation and reintroduction of DNA into cells or model organisms, usually to express a protein. The aim is to introduce new characteristics or attributes physiologically or physically, such as making a crop resistant to a herbicide, introducing a novel trait, enhancing existing ones, or producing a new protein or enzyme. Successful endeavours include the manufacture of human insulin through the use of modified bacteria, the manufacture of erythropoietin in Chinese hamster ovary cells, and the production of new types of experimental mice such as the OncoMouse (cancer mouse) for research.

Since a protein sequence is specified by a segment of DNA called a gene, novel versions of that protein can be produced by changing the DNA sequence of the gene. There are a number of ways through which this could be achieved. After isolating a section of DNA that includes the gene, the gene or required portion of the gene is cut out. After modification of the sequence if necessary, it may be introduced (spliced) into a different DNA segment or into a vector for transformation into living cells. Daniel Nathans and Hamilton Smith received the 1978 Nobel Prize in Physiology or Medicine for their isolation of restriction endonucleases, which are able to cut DNA at specific sites. Together with ligase, which can join fragments of DNA together, restriction enzymes formed the initial basis of recombinant DNA technology. Some groups have argued[citation needed] that genetic engineering is wrong and is "doing the work of God", but most scientists believe that genetic engineering is essential to help future medical discoveries[citation needed]. However, even with regard to this technology's great potential, scientists around the world have raised concerns about the introduction of genetically engineered plants and animals into the environment and the potential dangers of human consumption of GM foods. They say that these organisms have the potential to spread their modified genes into native populations thereby disrupting natural ecosystems. See also GM Food Controversies, and Genetically modified organism for more information on GM controversies. Professor Stephen Hawking defended the genetic enhancing of our species in order to compete with Artificial intelligence.

Applications
The first genetically engineered drug was human insulin, approved by the United States Food and Drug Administration in 1982. Another early application of genetic engineering was to create human growth hormone as replacement for a drug that was previously extracted from human cadavers. In 1986 the FDA approved the first genetically engineered vaccine for humans, for hepatitis B. Since these early uses of the technology in medicine, the use of GE has expanded to supply many drugs and vaccines.

One of the best known applications of genetic engineering is the creation of genetically modified organisms (GMOs).

There are potentially momentous biotechnological applications of GM, for example oral vaccines produced naturally in fruit, at very low cost.

A radical ambition of some groups is human enhancement via genetics, eventually by molecular engineering. See also: transhumanism.

Genetic engineering and research
Although there has been a tremendous revolution in the biological sciences in the past twenty years, there is still a great deal that remains to be discovered. The completion of the sequencing of the human genome, as well as the genomes of most agriculturally and scientifically important plants and animals, has increased the possibilities of genetic research immeasurably. Expedient and inexpensive access to comprehensive genetic data has become a reality with billions of sequenced nucleotides already online and annotated.

Now that the rapid sequencing of arbitrarily large genomes has become a simple, if not trivial affair, a much greater challenge will be elucidating function of the extraordinarily complex web of interacting proteins, dubbed the proteome, that constitutes and powers all living things. Genetic modification permits alteration of the primary structure of proteins and has therefore become a powerful tool in analyzing structure-function relationships in protein research. The use of the term "genetic engineering" to describe the experimental genetic modification of whole organisms, however, suggests a level of precision and an understanding of developmental biological principles beyond what has been achieved. Nonetheless, research progress has been made using a wide variety of techniques, including:

Loss of function, such as in a knockout experiment, in which an organism is engineered to lack the activity of one or more genes. This allows the experimenter to analyze the defects caused by this mutation, and can be considerably useful in unearthing the function of a gene. It is used especially frequently in developmental biology. A knockout experiment involves the creation and manipulation of a DNA construct in vitro, which, in a simple knockout, consists of a copy of the desired gene which has been slightly altered such as to cripple its function. The construct is then taken up by embryonic stem cells, where the engineered copy of the gene replaces the organism's own gene. These stem cells are injected into blastocysts, which are implanted into surrogate mothers. Another method, useful in organisms such as Drosophila (fruit fly), is to induce mutations in a large population and then screen the progeny for the desired mutation. A similar process can be used in both plants and prokaryotes. Gain of function experiments, the logical counterpart of knockouts. These are sometimes performed in conjunction with knockout experiments to more finely establish the function of the desired gene. The process is much the same as that in knockout engineering, except that the construct is designed to increase the function of the gene, usually by providing extra copies of the gene or inducing synthesis of the protein more frequently.

'Tracking' experiments, which seek to gain information about the localization and interaction of the desired protein. One way to do this is to replace the wild-type gene with a 'fusion' gene, which is a juxtaposition of the wild-type gene with a reporting element such as Green Fluorescent Protein (GFP) that will allow easy visualization of the products of the genetic modification. While this is a useful technique, the manipulation can destroy the function of the gene, creating secondary effects and possibly calling into question the results of the experiment. More sophisticated techniques are now in development that can track protein products without mitigating their function, such as the addition of small sequences which will serve as binding motifs to monoclonal antibodies. Expression studies aim to discover where and when specific proteins are produced. In these experiments the DNA sequence before the DNA that codes for a protein, known as a gene's promoter is reintroduced into an organism with the protein coding region replaced by a reporter gene such as GFP or an enzyme that catalyzes the production of a dye. Thus the time and place where a particular protein is produced can be observed. Expression studies can be taken a step further by altering the promoter to find which pieces are crucial for the proper expression of the gene and are actually bound by transcription factor proteins; this process is known as promoter bashing.

Recombinant DNA is a form of artificial DNA which is engineered through the combination or insertion of one or more DNA strands, thereby combining DNA sequences which would not normally occur together. In terms of genetic modification, recombinant DNA is produced through the addition of relevant DNA into an existing organismal genome, such as the plasmid of bacteria, to code for or alter different traits for a specific purpose, such as immunity. It differs from genetic recombination, in that it does not occur through processes within the cell or ribosome, but is exclusively engineered.

The Recombinant DNA technique was engineered by Stanley Norman Cohen and Herbert Boyer in 1973. They published their findings in a 1974 paper entitled "Construction of Biologically Functional Bacterial Plasmids in vitro", which described a technique to isolate and amplify genes or DNA segments and insert them into another cell with precision, creating a transgenic bacterium. Recombinant DNA technology was made possible by the discovery of restriction endonucleases by Werner Arber, Daniel Nathans, and Hamilton Smith, for which they received the 1978 Nobel Prize in Medicine.

Introduction
Because of the importance in DNA in the replication of new structures and characteristics of living organisms, it has widespread importance in recapitulating via viral or non-viral vectors, both desirable and undesirable characteristics of a species to achieve characteristic change or to counteract effects caused by genetic or imposed disorders that have effects upon cellular or organismal processes.Through the use of recombinant DNA, genes that are identified as important can be amplified and isolated for use in other species or applications, where there may be some form of genetic illness or discrepancy, and provides a different approach to complex biological problem solving

Applications and methods

Cloning and relation to plasmids
Cloning

A simple example of how a desired gene is inserted into a plasmid. In this example, the gene specified in the white color becomes useless as the new gene is added.The use of cloning is interrelated with Recombinant DNA in classical biology, as the term "clone" refers to a cell or organism derived from a parental organism, with modern biology referring to the term as a collection of cells derived from the same cell which remain identical. In the classical instance, the use of recombinant DNA provides the initial cell from which the host organism is then expected to recapitulate when it undergoes further cell division, with bacteria remaining a prime example due to the use of viral vectors in medicine which contain recombinant DNA inserted into a structure known as a plasmid.

Plasmids are extrachromosomal self replicating circular forms of DNA present in most bacteria, such as Escherichia coli (E. Coli), contain genes related to catabolism and metabolic activity, and allow the carrier bacterium to survive and reproduce in conditions present within other species and environments. These genes represent characteristics of resistance to bacteriophages and antibiotics and some heavy metals, but can also be fairly easily removed or separated from the plasmid by restriction endonucleases, which regularly produce "sticky ends" and allow the attachment of a selected segment of DNA, which codes for more "reparative" substances, such as peptide hormone medications including insulin, growth hormone, and oxytocin. In the introduction of useful genes into the plasmid, the bacteria is then used as a viral vector, which is encouraged to reproduce so as to recapitulate the altered DNA within other cells it infects and increase the amount of cells with the recombinant DNA present within them.

The use of plasmids is also key within gene therapy, where their related viruses are used as cloning vectors or carriers, which are means of transporting and passing on genes in recombinant DNA through viral reproduction throughout an organism. As a general definition of plasmids, the definition is that they contain three common features -- a replicator, selectable marker and a cloning site. The replicator or "ori" refers to the origin of replication with regards to location and bacteria where replication begins. The marker refers to a gene which usually contains resistance to an antibiotic, but may also refer to a gene which is attached alongside the desired one, such as that which confers luminescence to allow identification of successfully recombined DNA. The cloning site is a sequence of nucleotides representing one or more positions where cleavage by restriction endonucleases occurs. Most eukaryotes do not maintain canonical plasmids; yeast is a notable exception. In addition, the Ti plasmid of the bacterium Agrobacterium tumefaciens can be used to integrate foreign DNA into the genomes of many plants. Other methods of introducing or creating recombinant DNA in eukaryotes include homologous recombination and transfection with modified viruses.

Chimeric plasmids

An example of chimeric plasmid formation from two "blunt ends" via the enzyme, T4 Ligase.Main article: Chimeric DNA When recombinant DNA is then further altered or changed to host additional strands of DNA, the molecule formed is referred to as "chimeric" DNA molecule, with reference to the mythological chimera which consisted as a composite of several animals. The presence of chimeric plasmid molecules is somewhat regular in occurrence as throughout the lifetime of an organism the propagation by vectors ensures the presence of hundreds of thousands of organismal and bacterial cells which all contain copies of the original chimeric DNA.

In the production of chimeric plasmids, the processes involved can be somewhat uncertain as the intended outcome of the addition of foreign DNA may not always be achieved and may result in the formation of unusable plasmids. Initially, the plasmid structure is linearised to allow the addition by bonding of complimentary foreign DNA strands to single-stranded "overhangs" or "sticky ends" present at the ends of the DNA molecule from staggered, or "S shaped" cleavages produced by restriction endonucleases.

A common vector used for the donation of plasmids originally was the bacterium Escherichia coli and later, the EcoRI derivative which was used for it's versatility with addition of new DNA by "relaxed" replication when inhibited by chloramphenicol and spectinomycin; later being replaced by the pBR322 plasmid.In the case of EcoRI, the plasmid can anneal with the presence of foreign DNA via the route of sticky-end ligation, or with "blunt ends" via blunt-end ligation, in the presence of the phage T4 ligase , which forms covalent links between 3-carbon OH and 5-carbon PO4 groups present on blunt ends. Both sticky-end, or overhang ligation and blunt-end ligation can occur between foreign DNA segments, and cleaved ends of the original plasmid depending upon the restriction endonuclease used for cleavage.

A Vector DNA is a small piece of DNA containing regulatory and coding sequences of interest.

A vector is a DNA molecule into which foreign fragments of DNA may be inserted. A vector functions like a "molecular carrier", which will carry fragments of DNA into a host cell.

Vectors are usually derived from plasmids, which are small, circular, double-stranded DNA molecules occurring naturally in the cytoplasm of bacteria.

Vectors contain an origin of replication, which enables the vector, together with the foreign DNA fragment inserted into it, to replicate.

Vectors contain genetic markers that allow for selection of cells which have taken up the plasmid DNA.

Vector DNA functions to insert and amplify a gene into a target genome. Vector DNA can be used in a DNA vaccine.

Viral vectors are a tool commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism (in vivo) or in cell culture (in vitro). Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. Delivery of genes by a virus is termed transduction and the infected cells are described as transduced.

Molecular biologists first harnessed this machinery in the 1970s. Paul Berg used a modified SV40 virus containing DNA from the bacteriophage lambda to infect monkey kidney cells maintained in culture. Key properties of a viral vector Viral vectors are tailored to their specific applications but generally share a few key properties.

Safety. Although viral vectors are occasionally created from pathogenic viruses, they are modified in such a way as to minimize the risk of handling them. This usually involves the deletion of a part of the viral genome critical for viral replication. Such a virus can efficiently infect cells but, once the infection has taken place, requires a helper virus to provide the missing proteins for production of new virions. Low toxicity. The viral vector should have a minimal effect on the physiology of the cell it infects. Stability. Some viruses are genetically unstable and can rapidly rearrange their genomes. This is detrimental to predictability and reproducibility of the work conducted using a viral vector and is avoided in their design.

Cell type specificity. Most viral vectors are engineered to infect as wide a range of cell types as possible. However, sometimes the opposite is preferred. The viral receptor can be modified to target the virus to a specific kind of cell.

Applications
Basic research
Viral vectors were originally developed as an alternative to transfection of naked DNA for molecular genetic experiments. Compared to traditional methods such as calcium phosphate precipitation, transduction can ensure that nearly 100% of cells are infected without severely affecting cell viability. Furthermore, some viruses integrate into the cell genome facilitating stable expression. However, transfection is still the method of choice for many applications as construction of a viral vector is a much more laborious process. Protein coding genes can be expressed using viral vectors, commonly to study the function of the particular protein. Viral vectors, especially retroviruses, stably expressing marker genes such as GFP are widely used to permanently label cells to track them and their progeny, for example in xenotransplantation experiments, when cells infected in vitro are implanted into a host animal. Genes inserted into the vector can encode shRNAs and miRNAs used to efficiently block or silence production of a specific protein. Such knock-down experiments are much quicker and cheaper to carry out than gene knockout. But as the silencing is sometimes non-specific and has off-target effects on other genes, it provides less reliable results.

Gene therapy
Main article: Gene therapy
In the future gene therapy may provide a way to cure genetic disorders, such as severe combined immunodeficiency or cystic fibrosis. Several gene therapy trials have used viruses to deliver 'good' genes to the cells of the patient's body. There have been a huge number of laboratory successes with gene therapy. However, several problems of viral gene therapy must be overcome before it gains widespread use. Immune response to viruses not only impedes the delivery of genes to target cells but can cause severe complications for the patient. In one of the early gene therapy trials in 1999 this led to the death of Jesse Gelsinger, who was treated using an adenoviral vector.

Some viral vectors, for instance lentiviruses, insert their genomes at a seemingly random location on one of the host chromosomes, which can disturb the function of cellular genes and lead to cancer. In a severe combined immunodeficiency retroviral gene therapy trial conducted in 2002, two of the patients developed leukemia as a consequence of the treatment. Adeno-associated virus-based vectors are much safer in this respect as they always integrate at the same site in the human genome.

Vaccines
Viruses expressing pathogen proteins are currently being developed as vaccines against these pathogens, based on the same rationale as DNA vaccines. T-lymphocytes recognise cells infected with intracellular parasites based on the foreign proteins produced within the cell. T cell immunity is crucial for protection against viral infections and such diseases as malaria. A viral vaccine induces expression of pathogen proteins within host cells similarly to the Sabin Polio vaccine and other attenuated vaccines. However, since viral vaccines contain only a small fraction of pathogen genes, they are much safer and sporadic infection by the pathogen is impossible. Adenoviruses are being actively developed as vaccines.

Types of viral vectors

Retroviruses
Main article: Retroviruses
Retroviruses are the one of mainstays of current gene therapy approaches. The recombinant retroviruses such as the Moloney murine leukemia virus have the ability to integrate into the host genome in a stable fashion. They contain a reverse transcriptase which allows integration into the host genome. They have been used in a number of FDA-approved clinical trials such as the SCID-X1 trial.[4] The primary drawback to use of retroviruses such as the Moloney retrovirus involves the requirement for cells to be actively dividing for transduction. As a result, cells such as neurons are very resistant to infection and transduction by retroviruses. There is a concern for insertional mutagensis due to the integration into the host genome which can lead to cancer or leukemia.

Lentiviruses
Main article: Lentivirus
Packaging and infection by a lentiviral vector.Lentiviruses are a subclass of retroviruses. They are widely adapted as vectors thanks to their ability integrate into the genome of non-dividing as well as dividing cells. The viral genome in the form of RNA is reverse-transcribed when the virus enters the cell to produce DNA, which is then inserted into the genome at a random position by the viral integrase enzyme. The vector, now called a provirus, remains in the genome and is passed on to the progeny of the cell when it divides. The site of integration is unpredictable, which can pose a problem. The provirus can disturb the function of cellular genes and lead to activation of oncogenes promoting the development of cancer, which raises concerns for possible applications of lentiviruses in gene therapy.

For safety reasons lentiviral vectors never carry the genes required for their replication. To produce a lentivirus, several plasmids are transfected into a so-called packaging cell line, commonly HEK 293. One or more plasmids, generally referred to as packaging plasmids, encode the virion proteins, such as the capsid and the reverse transcriptase. Another plasmid contains the genetic material to be delivered by the vector. It is transcribed to produce the single-stranded RNA viral genome and is marked by the presence of the ? (psi) sequence. This sequence is used to package the genome into the virion.

Adenoviruses
Main article: Adenovirus
As opposed to lentiviruses, adenoviral DNA does not integrate into the genome and is not replicated during cell division. This limits their use in basic research, although adenoviral vectors are occasionally used in in vitro experiments. Their primary applications are in gene therapy and vaccination. Since humans commonly come in contact with adenoviruses, which cause respiratory, gastrointestinal and eye infections, they trigger a rapid immune response with potentially dangerous consequences. To overcome this problem scientists are currently investigating adenoviruses to which humans do not have immunity.

© Copyright 2008     ASLOC 22