Genetic engineering
From Activating Evolution
Genetic engineering, recombinant DNA technology, genetic modification/manipulation (GM) and gene splicing are terms that are applied to the direct manipulation of an organism's genes. Genetic modification of an organism can be achieved through a number of methods, most notably traditional breeding and recombinant technologies. The goal of both is the same, introduction of DNA (in the form of a gene) which in turn finds expression as favorable physical characteristics. Transgenic endeavors have found success in improving crop technology, 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. Some groups have argued 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. 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 (GMO) for more information on GM controversies. Professor Stephen Hawking defended the genetic enhancing of our species in order to compete with Artificial intelligence.
[edit] Methodology
There are a number of ways through which genetic engineering is accomplished. Essentially, the process has four main steps.
1) Isolation
2) Insertion into a vector
3) transformation
4) Tests to isolate genetically modified organism (GMO)
Isolation is achieved by identifying the gene of interest that the scientist wishes to insert into the organism, usually using existing knowledge of the various functions of genes. DNA information can be obtained from cDNA or gDNA libraries, and amplified using PCR techniques. If necessary, ie for insertion of eukaryotic genomic DNA into prokaryotes, further modification may be carried out such as removal of introns or ligating prokaryotic promoters.
Once the gene of interest is isolated, it is inserted into a vector such as a plasmid. Other vectors can also be used, such as viral vectors, and non-prokaryotic ones such as liposomes, or even direct insertion using DNA guns. Restriction enzymes and ligases are of great use in this crucial step if it is being inserted into prokaryotic or viral vectors. Daniel Nathans and Hamilton Smith received the 1978 Nobel Prize in Physiology or Medicine for their isolation of restriction endonucleases.
Once the vector is obtained, it can be used to transform the target organism. Depending on the vector used, it can be complex or simple. For example, using raw DNA with DNA guns is a fairly straightforward process but with low success rates, where the DNA is coated onto particles such as gold and fired directly into a cell. Other more complex methods, such as bacterial transformation or using viruses as vectors have higher success rates.
After transformation, the GMO can be isolated from those that have failed to take up the vector in various ways. One method is testing with DNA probes that can hybridize to the gene of interest that was supposed to have been inserted, another would be to package resistance genes along with the vector, such that the resulting GMO is resistant to certain chemicals, such as penicillin, and then they can be grown on agar dishes with penicillin, to ensure only those that have taken up the vector will survive.
[edit] 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) such as foods and vegetables that resist pest and bacteria infection and have longer freshness.
There are potentially momentous biotechnological applications of GM, for example oral vaccines produced naturally in fruit, at very low cost.
An ambition of some GE is human enhancement via genetics, eventually by molecular engineering. See also: transhumanism.
[edit] Genetic engineering and research
Although there has been a tremendous[1] 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.
- 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 (fruitfly), 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.
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