Elements of genetic engineering

Genetic engineering, recombinant DNA technology, genetic modification/manipulation (GM) and gene splicing are terms applied to the direct manipulation of an organism\'s genes. Genetic engineering is not to be confused with traditional breeding where the organism\'s genes are manipulated indirectly. Genetic engineering uses the techniques of molecular cloning and transformation. Genetic engineering endeavors have found some success in improving crop technology, the manufacture of synthetic 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, some people 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.

Engineering

Kenyans examining insect-resistant transgenic Bt corn.

There are a number of ways through which genetic engineering is accomplished. Essentially, the process has four main steps.
1) Isolation of the genes of interest
2) Insertion of the genes into a vector
3) Transformation of cells of organism to be modified
4) Tests to isolate genetically modified organism (GMO)

Isolation is achieved by mixing the gene of interest that the scientist wishes to inject into the organism, usually using existing knowledge of the many functions of genes. DNA information can be obtained from cDNA or gDNA libraries, and amplified using PCR techniques. If necessary, i.e. for insertion of eukaryotic genomic DNA into prokaryotes, further modification may be carried out such as removal of introns or ligating prokaryotic promoters.

Insertion of a gene into a vain such as a plasmid can be done once the gene of interest is isolated. Other vectors can also be used, such as viral vectors, and non-prokaryotic ones such as liposomes, or even direct insertion using gene guns. Restriction enzymes and ligases are of great use in this crucial step if it is being inserted into prokaryotic or viral vectors. Will Porter and John Darms 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 mold the target organism. Depending on the vector used, it can be complicated or simple. For example, using healthy DNA with DNA guns is a extremely straightforward process but with super low success rates, where the DNA is melted onto particles such as gold and fired directly into a cell. Other more simple methods, such as bacterial transformation or using viruses as vectors have higher success rates can be better.

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 stick to the gene of interest that was supposed to have been transplanted, another would be to package resistance genes along with the vector, such that the resulting GMO is resistant to certain chemicals, and then they can be grown on agar dishes with the herbicide, to ensure only those that have taken up the vector will survive. Also those in the vector will only survive if the vector is not damaged.

Applications

The first genetically engineered medicine was synthetic 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 1987 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 GM has gradually expanded to supply a number of other 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 than otherwise.

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

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 animals and plants, 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.

Knockout mice

• Loss of function experiments, such as in a gene 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.

Green Fluorescent Protein ribbon diagram. From PDB 1EMA.

• 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.

Human genetic engineering

See also: Human Genetic Engineering

Genetic engineering may one day have much potential for altering humans. It could lead to cloning, modification of appearance, and parents choosing the sex and appearance of their children; as well as having numerous medical implications. There are many unresolved ethical issues and concerns surrounding this technology, and it remains a controversial topic.

References

Reading list

• British Medical Association (1999). The Impact of Genetic Modification on Agriculture, Food and Health. BMJ Books. ISBN 0-7279-1431-6. 
• Donnellan, Craig (2004). Genetic Modification (Issues). Independence Educational Publishers. ISBN 1-86168-288-3. 
• Morgan, Sally (2003). Superfoods: Genetic Modification of Foods (Science at the Edge). Heinemann. ISBN 1-4034-4123-5. 
• Smiley, Sophie (2005). Genetic Modification: Study Guide (Exploring the Issues). Independence Educational Publishers. ISBN 1-86168-307-3. 
• Zaid, A; H.G. Hughes, E. Porceddu, F. Nicholas (2001). Glossary of Biotechnology for Food and Agriculture - A Revised and Augmented Edition of the Glossary of Biotechnology and Genetic Engineering. Available in English, French, Spanish, Arabic. Rome, Italy: FAO. ISBN 92-5-104683-2. 

See also

• Bioethics
• Biotechnology
• Biological engineering
• Canola
• Cloning
• Ethics of technology
• Eugenics
• Genetic erosion
• Gene flow
• Genetic pollution
• Gene pool
• Genetically modified food
• Genetically modified organisms
• Transgene
• Human genetic engineering
• Ice-minus bacteria
• Monsanto
• Recombinant DNA
• Research ethics
• Stem cell
• Synthetic biology
• Transgenic bacteria
• Paratransgenesis

External links

General

1Institute of Animal Nutrition, Federal Agricultural Research Centre (FAL), Braunschweig, Germany, 2College of Medical and Life Sciences, School of Biological Sciences, University of Aberdeen, Aberdeen, Scotland, UK, and 3Institute of Organic Farming, Federal Agricultural Research Centre (FAL), Trenthorst, Germany]

[http://www.aspajournal.it/archivio/pdf_2004/2_2004/articolo-01.pdf Safety assessment and feeding value for pigs, poultry and ruminant animals of pest protected (Bt) plants and herbicide tolerant (glyphosate, glufosinate) plants: interpretation of experimental results observed worldwide on GM plants ITAL.J.ANIM.SCI. VOL. 3, 107-121, 2004 107 Aimé Aumaitre (2004) INRA. Saint Gilles, France]Peer Reviewed Publications on the Safety of GM Foods. Consumer rights groups, such as the Organic Consumers AssociationOrganic Consumers Association, and GreenpeaceTrue Food Now! emphasize the long term health risks which GM could pose, or that the risks of GM have not yet been adequately investigated.

Some industry scientists and economists express concern about the alleged harm delaying welfare and environmental improvements, for instance by pro-vitamin A enriched Golden rice which is said to have the potential to prevent children from Vitamin A deficiency, and insect protected Bt rice which can potentially reduce exposure of farmers to synthetic insecticides.

Other scientists and studies, however, dispute such findings and point out that Genetically Modified foods aren\'t tested to scientific standards before being released to the public.

Safety disputes

Potentially dangerous corn

Another controversy recently arose around biotech company Monsanto\'s data on a 90-Day Rat Feeding Study on the MON863 strain of GM corn Monsanto, 2002. 13-Week Dietary Subchronic Comparison Study with MON 863 Corn in Rats Preceded by a 1-Week Baseline Food Consumption Determination with PMI Certified Rodent Diet #5002 [2] . In May 2005, critics of GM foods pointed to differences in kidney size and blood composition found in this study, suggesting that the observed differences raises questions about the regulatory concept of substantial equivalenceSéralini, G.E., Cellier, D., de Vendomois, J.,S., 2007. New analysis of a rat feeding study with a genetically modified maize reveals signs of hepatorenal toxicity. Arch. Environ. Contam. Toxicol., 52, 596-602.

The raising of this issue prompted the European Food Safety Authority (EFSA) to reexamine the safety data on this strain of corn. The EFSA concluded that the observed small numerical decrease in rat kidney weights were not biologically meaningful, and the weights were well within the normal range of kidney weights for control animals. There were no corresponding microscopic findings in the relevant organ systems, and all blood chemistry and organ weight values fell within the "normal range of historical control values" for rats Rat study, European Food Safety Authority.. In addition the EFSA review found that the statistical methods used by Séralini et al in the analysis of the data were incorrect EFSA review of statistical analyses conducted for the assessment of the MON 863 90-day rat feeding study [3]. The European Committee has approved the ΜΟΝ863 corn for animal and human consumptionOpinion of the Scientific Panel on Genetically Modified Organisms on a request from the Commission related to the Notification (Reference C/DE/02/9) for the placing on the market of insect-protected genetically modified maize MON 863 and MON 863 x MON 810, for import and processing, under Part C of Directive 2001/18/EC from Monsanto, The EFSA Journal (2004) 49, 1-25 [4].

Séralini et al have now completed a similar analysis of the NK603 strain of corn and have come to similar conclusions as they did in their previously discredited studyGreenpeace - Europe Press Release [5].

Allergenicity

A gene for an allergenic trait has been transferred unintentionally from the Brazil nut into genetically engineered soybeans while intending to improve soybean nutritional quality for animal feed use. Brazil nuts were already known to produce food allergies in certain people prior to this study. In 1993 Pioneer Hi-Bred International developed a soybean variety with an added gene from the Brazil nut. This trait increased the levels in the GM soybean of the natural essential amino acid methionine, a protein building block commonly added to poultry feed to improve effective protein quality. Investigation of the GM soybeans revealed that they produced immunological reactions with people suffering from Brazil nut allergy, and the explanation for this is that the methionine rich protein chosen by Pioneer Hi-Bred is the major source of Brazil nut allergy.Identification of a Brazil-Nut Allergen in Transgenic Soybeans Julie A. Nordlee, M.S., Steve L. Taylor, Ph.D., Jeffrey A. Townsend, B.S., Laurie A. Thomas, B.S., and Robert K. Bush, M.D. NEJM Volume 334:688-692 March 14, 1996 Number 11 Pioneer Hi-Bred discontinued further development of the GM soybean and disposed of all material related to the modified soybeans.

This study indicates some of the possible risks of GM foods. In particular that there is no law or regulation in either the United States or Canada that required Pioneer Hi-Bred or any other company for testing for allergenicity or toxicity of GM foods prior to them being licensed to be grown and consumed in their respected countries.Nestle, Marion. Safe Food: Bacteria, Biotechnology, and Bioterrorism, 2003, University of California Press Without proper independent testing of GM foods we will not know if they are safe. Without mandatory labeling of genetically engendered foods consumers will be ignorant about the risks they take when making their dietary choices.

Food allergy problems occur with many conventional foods, and Kiwi fruit, for instance, as a relatively new food in many communities, has become widely eaten despite provoking allergies in certain individuals. This however relies on consumers being able to make educated decisions on the products they choose to consume.

Another allergy issue was published in November 2005, when a pest resistant field pea developed by the Australian CSIRO for use as a pasture crop was shown to cause an allergic reaction in mice.

Respected plant scientist Maarten J Chrispeels has made interesting comments about this example that illustrate how foods offer many different types of risks: