The genome of both animals and plants has been altered for quite some time through various traditional breeding methods and organisms with favourable characteristics have being artificially selected to breed successive generations. These procedures, however, have been limited by naturally occurring variations. Recent advances in genetic engineering have managed to get easy for scientists to precisely control any genetic changes introduced into an organism. Genes in one species can, through genetic engineering, be incorporated into an completely different species.
Any organism which includes had its genetic material altered is defined as a genetically modified organism (GMO). The definition states that a GMO includes "any living organism that possesses a novel combo of genetic material obtained through the use of modern biotechnology". It is important to note that regarding humans, even if indeed they have had their genes altered as a result of, gene remedy for instance, they are not considered to be GMOs.
A major purpose of the field of genetics is to classify genes according with their function. The scientific study of genes in living organisms can be separated into three different strategies. The first two, which analyse natural variation and random mutagenesis, will be the primary ways of 'forward genetics' where the genotype associated with an abnormal phenotype is studied. Regarding random mutagenesis, it is often difficult to trace a phenotype back to a particular gene as much chromosomal loci are simultaneously targeted.
Thus a third kind of research strategy, 'reverse genetic' techniques, has been developed before three decades. This technique, in which a specific gene is modified and the phenotype is subsequently investigated, provided tools for the study of gene function in a targeted manner. Among the most commonly used animals are Drosophila melanogaster (fruit fly), Caenorhabditis elegans (nematode) and Mus musculus (house mouse), and plants Nicotiana tabacum (tobacco), Arabidopsis thaliana (thale cress) and Triticum aestivum (common wheat) that have each been essential for the identification of genes implicated in aging, cell differentiation, development, and other significant biological functions. Transgenic rats are of great importance in neuroscience as they have been extensively used in behavioural paradigms (Abbott 2004) while recently, the first transgenic primate disease model, for Huntington's disease, was created (Yang et al. 2008).
Research has progressed through the use of various techniques. One of these techniques is the increased loss of function method where an organism is modified so that one or more genes lose their activity. This technique has allowed researchers to analyze defects caused by particular mutations and has been extremely useful in identifying the function of an gene. Another method is the knockout experiment which involves the creation and manipulation of the DNA construct in vitro. In a simple knockout, this construct contains a copy of the required gene which includes been slightly changed to lose its function. The modified gene is then taken up by embryonic stem cells, and it replaces the organism's own gene. These stem cells are then injected into a blastocyst which is implanted into a surrogate mother.
Furthermore, the gain of function method is generally performed with the knockout method so that the function of any desired gene can be more finely identified. The procedure is very similar to knockout engineering, however in this case the construct amplifies the function of the gene, with the addition of extra copies of the gene or inducing more frequent synthesis of the protein.
Information on the localization and interaction of any protein can be obtained through 'tracking' experiments. In this technique the wild-type gene is replaced with a 'fusion' gene, which is a combo of the wild-type gene with a reporting component such as Green Fluorescent Protein (GFP). The reporting factor allows visualization of the merchandise of the modification. Moreover, expression studies can display the time and location of the synthesis of the protein. In this system the promoter is reintroduced into an organism however the coding region is replaced by a reporting factor or an enzyme catalyzing the production of a dye. An additional advancement in expression studies has been the process promoter bashing, where the promoter is altered so as to find which pieces are crucial for proper gene expression and are bound by transcription factor proteins.
The use of these, and other, methods to investigate critical questions in genetics has become standard practice. In particular, GMOs have been needed for 1) Identifying a gene's function and any molecular elements related to it; 2) Creating models of human diseases; 3) Determining and confirming drug targets and specificity and 4) Investigating chronological aspects of gene function.
1) Identifying a gene's function and any molecular elements related to it
Targeted mutagenesis has been used extensively in the field of neuroscience. One of the initial groundbreaking experiments using targeted mutagenesis was completed by Eric Kandel at Columbia University. Utilizing the tetracycline inducible system, they expressed a calcium-independent form of the calcium dependent kinase, calcium-calmodulin kinase II (CaMKII), which is specific for the forebrain. In addition they observed a decline in spatial memory and hippocampal long-term potentiation (LTP). This major breakthrough displayed how both temporal and spatial control over molecular elements can help to identify the function of a specific gene and its own role in brain function.
Plant modification techniques have been invaluable tools for genetic research. Through the use of these methods, geneticists have gained detailed knowledge on the function of specific plant genes, their expression and the properties of the proteins they encode. One important trait that is studied using transgenic plants is plant mechanical strength, which has an agronomic significance. Transgenic rice from the Indica variety was used to elucidate the molecular mechanism controlling the mechanical strength of crops. Mutations on the BC1 gene, which is expressed mostly in developing sclerenchyma cells and vascular bundles, were introduced into rice and led to a loss of cell wall thickness and cellulose content and a rise in lignin level. This suggested that BC1 has a essential role in the biosynthesis of the cell walls of mechanical tissues.
2) Creating models of human diseases
Another significant use for GMOs is the development of models for human disorders. This can be done by introducing a mutated gene or eliminating a gene that includes a putative role in the disease. GMOs have been tremendously useful in modelling a wide range of illnesses, especially in neuro-scientific neuroscience. These include Alzheimer's disease (AD), Huntington's disease, cerebral ischemia and neuropsychiatric disorders. AD, for instance, is characterized by the presence of neurofibrillary tangles formed by hyper-phosphorylated tau protein and by amyloid ‹†-peptide (A‹†) plaques. Mutations in the amyloid precursor protein (APP), apolipoprotein E (APOE) and presenilin 1 and 2 (PS1, PS2), have all been proven to truly have a role in the condition. Research on transgenic mice has indicated an overexpression of APP and PS1 causes A‹† plaque formation and causes memory deficits, that are both symptoms of AD. The significant role of GMOs in testing potential causal mechanisms of human disease is consequently highlighted.
Recently, researchers in the University of Western Ontario dealing with scientists in Brazil used a distinctive transgenic mouse line to discover a previously unknown mechanism causing heart failure. The analysis showed that if the release of the neurotransmitter acetylcholine, which decreases cardiac activity, is reduced the probability of heart failure increases (ScienceDaily, 2010).
Fundamental discoveries made first in plants have been central inside our understanding of human biology, specifically cells, genes, molecular chaperones, transposable elements, programmed cell death, and gaseous hormones. Research on genetically modified A. thaliana has helped in the identification of genes involved in human disease. Scientists show that eighty-eight genes on chromosome 5 of an. thaliana are incredibly like the 289 genes associated with human disease syndromes which were established for comparison with D. melanogaster. As nearly all these are also greatly conserved between D. melanogaster and C. elegans, A. thaliana biology can be modified and used as a model to increase our understanding of human disease. Many of these genes encode proteins which have a conserved function. For instance, DNA excision repair genes (linked to xeroderma pigmentosum) and ATP-dependent copper transporters (associated with Wilson's and Menke's disease). Inside the latter the A. thaliana homologue is more similar to the human homologue than to the D. melanogaster or C. elegans counterparts.
3) Determining and confirming drug targets and specificity
The development of genetic models for diseases has greatly assisted in drug discovery and in identifying drug targets. Genetic modification can be extremely specific, for instance removing a gene which codes for a particular receptor subtype. This specificity ensures almost complete selectivity which is thus preferred to classical pharmacological approaches. For instance, by using both the knock-in and knockout methods scientists can examine the function of receptor subtypes. Specifically, transgenic mice have been used to establish the purpose of specific GABA receptor subunits in the diverse actions of diazepam. Diazepam, a benzodiazepine, acts on GABAA receptors which contain ‹±1-, ‹±2-, ‹±3-, or ‹±5- subunits. The mice where modified to carry point mutations in the benzodiazepine receptors of each of the subunits, and the investigators were thus able to genetically separate the distinct functions of diazepam (e. g. sedative or anxiolytic) acting at otherwise similar GABAA receptors.
Plants are incredibly significant as novel therapeutic drug leads. Nicotine, the main biologically active compound in N. tabacum, binds to the nicotinic acetylcholine receptors (nicAChRs) in the central nervous system (CNS) to produce a wide variety of biological effects. The nicAChR family consists of receptors made of ‹†-subunits and a series of structurally varied ‹±-subunits. The distinctions in the ‹±-subunit amino acids and in the subunit composition create variations in the receptor's binding site and present the members of the receptor family unique drug specificity. By using transgenic N. tabacum it was demonstrated a significant number of the plants contained compounds that are selective for brain receptors from the a7 nicAChR. Compounds which have this selectivity are of particular interest as drugs that act on these receptors may be useful to treat degenerative brain diseases such as AD.
4) Determining and confirming drug targets and specificity
In addition, GMOs have been used to analyse second messenger signaling pathways and also to determine vital developmental timing of gene function. The latter was demonstrated by using a tetracycline inducible knock-out of the serotonin 1A receptor. When the receptor was knocked out during development, it caused the knockout mouse to experience increased anxiety. If the receptor was knocked out in adults, however, the anxiety levels were normal. It was thus shown that the 1A receptor is associated to a developmental factor necessary for normal emotional behaviour.
Transgenic plants have also been effectively used to analyze regulated gene expression, as the expression can be investigated at various stages and in various tissues. The environmental factors that influence gene expression and which have been studied in transgenic plants include temperature, light intensity, anaerobic stress, and wounding. The introduction of flowers requires the collaboration of specialized tissues. Research was carried out on transgenic petunia to look for the genes controlling the development and differentiation in the flower. By analysing the 5-enolpyruvylshikimate-3-phosphate synthase gene, which is situated in high concentrations in flowers, the researchers could actually identify an upstream region that is accountable for the tissue-specific and developmentally regulated expression.
In summary, this technology has played a significant role in genetics. The most common applications of GMOs in neuro-scientific genetics have been highlighted: dissecting biological mechanisms, modelling human diseases, discovering and validating pharmaceuticals, and investigating crucial time windows in gene function. While there are many other types of approaches for creating GMOs and a great many other uses for these organisms, the role of GMOs in genetics has being the main focus, as the effect on this field has been substantial. New approaches to creating and using GMOs are continuously being developed, including adjustments and combinations of the discussed techniques, that may surely further impact genetics.
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