11 July 2008

Genetic engineering

Genetic engineering (genetic engineering) is a set of methods and technologies, including technologies for obtaining recombinant ribonucleic and deoxyribonucleic acids, for isolating genes from the body, manipulating genes and introducing them into other organisms [1].

Genetic engineering is an integral part of modern biotechnology, its theoretical basis is molecular biology, genetics. The essence of the new technology is directed, according to a predetermined program, the construction of molecular genetic systems outside the body (in vitro), followed by the introduction of the created structures into a living organism. As a result, their inclusion and activity is achieved in this organism and in its offspring. The possibilities of genetic engineering – genetic transformation, transfer of foreign genes and other material carriers of heredity into the cells of plants, animals and microorganisms, obtaining genetically engineered (genetically modified, transgenic) organisms with new unique genetic, biochemical and physiological properties and traits, make this direction strategic.

From the point of view of methodology, genetic engineering combines fundamental principles (genetics, cell theory, molecular biology, systems biology), achievements of the most modern postgenomic sciences: genomics, metabolomics, proteomics with real achievements in applied areas: biomedicine, agrobiotechnology, bioenergetics, biopharmacology, bioindustry, etc.

Genetic engineering belongs (along with biotechnology, genetics, molecular biology, and a number of other life sciences) to the field of natural sciences.

Historical background

Genetic engineering appeared thanks to the work of many researchers in various fields of biochemistry and molecular genetics. In 1953, J. Watson and F. Crick created a double–chiral DNA model, at the turn of the 50 - 60s of the 20th century the properties of the genetic code were clarified, and by the end of the 60s its universality was confirmed experimentally. There was an intensive development of molecular genetics, the objects of which were E.coli, its viruses and plasmids. Methods of isolation of highly purified preparations of intact DNA molecules, plasmids and viruses have been developed. The DNA of viruses and plasmids was injected into cells in a biologically active form, ensuring its replication and expression of the corresponding genes. In 1970, G. Smith was the first to isolate a number of restriction enzymes suitable for genetic engineering purposes. G. Smith found that the purified enzyme HindII obtained from bacteria retains the ability to cut nucleic acid molecules (nuclease activity) characteristic of living bacteria. The combination of DNA-restrictases (for cutting DNA molecules into certain fragments) and DNA–ligase enzymes isolated back in 1967 (for "stitching" fragments in an arbitrary sequence) can rightfully be considered a central link in the technology of genetic engineering.

The birth date of genetic engineering can be considered 1972, when P. Berg, S. Cohen, H. Boyer and colleagues (Stanford University) created the first recombinant DNA containing DNA fragments of the SV40 virus, bacteriophage and E. Coli.

Thus, by the beginning of the 70s, the basic principles of the functioning of nucleic acids and proteins in a living organism were formulated and theoretical prerequisites for genetic engineering were created

Academician A.A. Baev was the first scientist in our country who believed in the prospects of genetic engineering and led research in this field. Genetic engineering (by its definition) is the construction in vitro of functionally active genetic structures (recombinant DNA), or otherwise – the creation of artificial genetic programs.

Methods of genetic engineering

It is well known that traditional breeding has a number of restrictions that prevent the production of new animal breeds, plant varieties or races of practically valuable microorganisms:

1. absence of recombination in unrelated species. There are rigid barriers between species that make natural recombination difficult.
2. the inability to control the process of recombination in the body from the outside. The lack of homology between chromosomes leads to the inability to converge and exchange individual sites (and genes) during the formation of germ cells. As a result, it becomes impossible to transfer the necessary genes and ensure an optimal combination in a new organism of genes obtained from different parental forms;
3. the inability to accurately define the characteristics and properties of the offspring, because the recombination process is statistical.

The natural mechanisms that guard the purity and stability of the genome of an organism are almost impossible to overcome by the methods of classical breeding.

The technology of obtaining genetically modified organisms (GMOs) fundamentally solves the issues of overcoming all natural and interspecific recombination and reproductive barriers. Unlike traditional breeding, during which the genotype is changed only indirectly, genetic engineering allows you to directly interfere with the genetic apparatus, using the technique of molecular cloning. Genetic engineering allows you to operate with any genes, even those synthesized artificially or belonging to unrelated organisms, transfer them from one species to another, combine them in any order.

The technology includes several stages of creating GMOs:

1. Obtaining an isolated gene.
2. The introduction of a gene into a vector for embedding in the body.
3. Transfer of a vector with a construction into a modifiable recipient organism.
4. Molecular cloning.
5. Selection of GMOs.

The first stage – synthesis, isolation and identification of target DNA or RNA fragments and regulatory elements is very well developed and automated. An isolated gene can also be obtained from a phage library.

The second stage is the creation in vitro (in vitro) of a genetic construct (transgene), which contains one or more DNA fragments (encoding the sequence of amino acids of proteins) in combination with regulatory elements (the latter ensure the activity of transgenes in the body). Next, transgenes are embedded in DNA vectors for cloning using genetic engineering tools – restrictases and ligases. For the discovery of restrictases, Werner Arber, Daniel Nathans and Hamilton Smith were awarded the Nobel Prize (1978). As a rule, plasmids are used as a vector – small ring DNA molecules of bacterial origin.

The next stage is actually "genetic modification" (transformation), i.e. the transfer of the "vector – embedded DNA" construct into individual living cells. The introduction of a finished gene into the hereditary apparatus of plant and animal cells is a complex task that was solved after studying the features of the introduction of foreign DNA (virus or bacteria) into the genetic apparatus of the cell. The transfection process was used as the principle of introducing genetic material into the cell.

If the transformation was successful, then after effective replication, many daughter cells containing an artificially created genetic construct arise from one transformed cell. The basis for the appearance of a new trait in an organism is the biosynthesis of proteins new to the body – products of a transgene, for example, plants resistant to drought or insect pests in GM plants.

For unicellular organisms, the process of genetic modification is limited to embedding a recombinant plasmid with subsequent selection of modified descendants (clones). For higher multicellular organisms, for example, plants, it is mandatory to include the structure in the DNA of chromosomes or cellular organelles (chloroplasts, mitochondria) with subsequent regeneration of the whole plant from a separate isolated cell on nutrient media. In the case of animals, cells with an altered genotype are injected into the blastocides of the surrogate mother. The first GM plants were obtained in 1982 by scientists from the Institute of Plant Science in Cologne and Monsanto.

Main directions

The post-genomic era in the first decade of the XXI century raised the development of genetic engineering to a new level. The so-called Cologne Protocol "Towards Knowledge-Based Bioeconomics" [2] defined bioeconomics as "the transformation of life sciences knowledge into new, sustainable, environmentally efficient and competitive products". The roadmap of genetic engineering contains a number of areas: gene therapy, bioindustry, technologies based on animal stem cells, GM plants, GM animals, etc.

Genetically modified plants

There are various ways to introduce foreign DNA into plants.

For dicotyledonous plants, there is a natural vector for horizontal gene transfer: plasmids of agrobacteria. As for monocots, although in recent years certain successes have been achieved in their transformation by agrobacterial vectors, nevertheless, such a path of transformation meets significant difficulties.

For the transformation of plants resistant to agrobacteria, methods of direct physical transfer of DNA into the cell have been developed, they include: microparticle bombardment or ballistic method; electroporation; polyethylene glycol treatment; DNA transfer as part of liposomes, etc.

After the transformation of plant tissue in one way or another, it is placed in vitro on a special medium with phytohormones that promotes cell reproduction. The medium usually contains a selective agent against which transgenic, but not control cells acquire resistance. Regeneration most often goes through the callus stage, after which, with the right selection of media, organogenesis (shoot formation) begins. The formed shoots are transferred to the rooting medium, often also containing a selective agent for more rigorous selection of transgenic individuals.

The first transgenic plants (tobacco plants with embedded genes from microorganisms) were obtained in 1983. The first successful field trials of transgenic plants (resistant to viral infection of tobacco plants) were conducted in the USA already in 1986.

After passing all the necessary tests for toxicity, allergenicity, mutagenicity, etc., the first transgenic products appeared on sale in the USA in 1994. These were Flavr Savr tomatoes with delayed ripening, created by Calgen, as well as herbicide-resistant soy from Monsanto. Within 1-2 years, biotech firms put on the market a number of genetically modified plants: tomatoes, corn, potatoes, tobacco, soybeans, rapeseed, zucchini, radish, cotton.

In the Russian Federation , the possibility of obtaining transgenic potatoes by bacterial transformation using Agrobacterium tumefaciens was shown in 1990 .

Currently, hundreds of commercial firms around the world with a combined capital of more than $ 100 billion are engaged in obtaining and testing genetically modified plants. Plant genetic engineering biotechnology has already become an important industry for the production of food and other useful products, attracting significant human resources and financial flows.

In Russia, under the leadership of Academician K.G. Scriabin (Bioengineering Center of the Russian Academy of Sciences), GM potato varieties Elizaveta plus and Lugovskaya plus resistant to the Colorado potato beetle were obtained and characterized. According to the results of the inspection by the Federal Service for Supervision of Consumer Rights Protection and Human Well-Being on the basis of the expert opinion of the State Research Institute of Nutrition of the Russian Academy of Medical Sciences, these varieties have been state registered, entered into the state register and allowed for import, manufacture and turnover in the territory of the Russian Federation.

These GM potato varieties are fundamentally different from the usual ones by the presence in its genome of an embedded gene that determines 100% crop protection from the Colorado potato beetle without the use of any chemicals.

The first wave of transgenic plants approved for practical use contained additional resistance genes (to diseases, herbicides, pests, spoilage during storage, stress).

The current stage of the development of plant genetic engineering has been called "metabolic engineering". At the same time, the task is not so much to improve certain existing qualities of the plant, as with traditional breeding, as to teach the plant to produce completely new compounds used in medicine, chemical production and other fields. These compounds can be, for example, special fatty acids, useful proteins with a high content of essential amino acids, modified polysaccharides, edible vaccines, antibodies, interferons and other "medicinal" proteins, new polymers that do not pollute the environment and much, much more. The use of transgenic plants makes it possible to establish large-scale and cheap production of such substances and thereby make them more accessible for widespread consumption.

Genetically modified animals

Animal cells differ significantly from bacterial cells in their ability to absorb foreign DNA, therefore, methods and methods of introducing genes into embryonic cells of mammals, flies and fish remain in the focus of attention of genetic engineers.

The most genetically studied mammal is mice. The first success dates back to 1980, when D. Gordon and his colleagues demonstrated the possibility of introducing and integrating foreign DNA into the genome of mice. Integration was stable and persisted in the offspring. The transformation is performed by microinjection of cloned genes into one or both pronuclei (nuclei) of a newly embryo at the stage of a single cell (zygote). The male pronucleus introduced by the sperm is more often chosen, since its size is larger. After injection, the egg is immediately implanted into the oviduct of the adoptive mother, or given the opportunity to develop in culture to the blastocyst stage, after which it is implanted into the uterus.

Thus, human interferon and insulin genes, rabbit β-globin gene, herpes simplex virus thymidine kinase gene and mouse leukemia virus cDNA were injected. The number of molecules injected per injection ranges from 100 to 300,000, and their size ranges from 5 to 50 kb. Usually 10-30% of eggs survive, and the proportion of mice born from transformed eggs varies from several to 40%. Thus, the real efficiency is about 10%.

Genetically engineered rats, rabbits, sheep, pigs, goats, calves and other mammals were obtained by this method. Pigs carrying the somatotropin gene have been obtained in our country. They did not differ in growth rates from normal animals, but the change in metabolism affected the fat content. In such animals, lipogenesis processes were inhibited and protein synthesis was activated. The incorporation of insulin-like factor genes also led to a change in metabolism. GM pigs were created to study the chain of biochemical transformations of the hormone, and the side effect was to strengthen the immune system.

The most powerful protein-synthesizing system is found in breast cells. If you put the genes of foreign proteins under the control of a casein promoter, then the expression of these genes will be powerful and stable, and the protein will accumulate in milk. With the help of bioreactor animals (transgenic cows), milk has already been obtained, which contains the human protein lactoferrin. This protein is planned to be used for the prevention of gastroenterological diseases in people with low immunoresistance: AIDS patients, premature infants, cancer patients who have undergone radiotherapy.

An important area of transgenosis is the production of disease–resistant animals. The interferon gene related to protective proteins was embedded in various animals. Transgenic mice gained resistance – they did not get sick or hurt a little, but no such effect was found in pigs.

Application in scientific research

Gene knockout is a technique for removing one or more genes, which allows you to study the functions of a gene. To obtain knockout mice, the resulting genetically engineered structure is injected into embryonic stem cells, where the structure undergoes somatic recombination and replaces the normal gene, and the modified cells are implanted into the blastocyst of the surrogate mother. In a similar way, a knockout is obtained in plants and microorganisms.

Artificial expression is the addition of a gene to an organism that it previously did not have, also for the purpose of studying the function of genes. Visualization of gene products – used to study the localization of a gene product. Replacement of a normal gene with a constructed gene fused with a reporter element (for example, with a green fluorescent protein gene) provides visualization of the product of gene modification.

Investigation of the mechanism of expression. A small section of DNA located in front of the coding region (promoter) and serving to bind transcription factors is injected into the body, putting after it a reporter gene instead of its own, for example, GFP, catalyzing an easily detectable reaction. In addition to the fact that the functioning of the promoter in certain tissues becomes clearly visible at one time or another, such experiments make it possible to study the structure of the promoter by removing or adding DNA fragments to it, as well as artificially enhance gene expression.

Biosafety of genetic engineering activities

Back in 1975, scientists around the world at the Asilomar Conference raised the most important question: will the appearance of GMOs have a potentially negative impact on biological diversity? From that moment, simultaneously with the rapid development of genetic engineering, a new direction began to develop — biosafety. Its main task is to assess whether the use of GMOs has an undesirable impact on the environment, human and animal health, and the main goal is to open the way to the use of modern biotechnology achievements, while guaranteeing safety.

The biosafety strategy is based on a scientific study of the characteristics of GMOs, the experience of handling it, as well as information about its intended use and the environment into which it will be introduced. The joint long-term efforts of international organizations (UNEP, WHO, OECD), experts from different countries, including Russia, have developed basic concepts and procedures: biological safety, biological hazard, risk, risk assessment. Only after the full cycle of inspections has been successfully carried out, a scientific conclusion on the biosafety of GMOs is prepared. In 2005 WHO has published a report according to which the use of GM plants registered as food is also safe as their traditional counterparts.

How is biosafety ensured in Russia? The ratification of the Convention on Biodiversity in 1995 can be considered the beginning of Russia's inclusion in the global biosafety system. From that moment, the formation of the national biosafety system began, the starting point of which was the entry into force of the Federal Law of the Russian Federation "On State Regulation in the Field of Genetic Engineering" (1996). The Federal Law establishes the basic concepts and principles of state regulation and control of all types of work with GMOs. The Federal Law sets risk levels depending on the type of GMOs and the type of work, defines closed and open systems, the release of GMOs, etc.

Over the past years, one of the toughest regulatory systems has been formed in Russia. It is an extraordinary fact that the system of state regulation of GMOs started proactively, in 1996, before real genetically engineered organisms were declared for commercialization on the territory of Russia (the first GMO - GM soy – was registered for food use in 1999). The basic legal instruments are the state registration of genetically engineered modified organisms, as well as products derived from them or containing them, intended for use as food and feed.

To understand the current situation, it is important that during the 25 years that have passed since the first release of GM plants to the market, not a single significant negative impact on the environment and human and animal health has been revealed either during testing or during commercial use. Only one of the world's sources – the report of the authoritative AGBIOS society "Essential Biosafety" contains more than 1,000 references[3] to studies proving that food and feed obtained from biotechnological crops are as safe as traditional products are safe. However, to date, there is no regulatory framework in Russia that would allow the release of GM plants into the environment on the territory of our country, as well as products derived from them or containing them. As a result, as of 2010, not a single GM plant is grown on the territory of the Russian Federation for commercial purposes.

According to the forecast, according to the Cologne Protocol (2007), by 2030 the attitude towards agricultural GM crops will change towards approval of their use.

Achievements and development prospects

Genetic engineering in medicine

The needs of healthcare, the need to solve the problems of population aging form a steady demand for genetically engineered pharmaceuticals (with annual sales of $ 26 billion) and medical and cosmetic products from plant and animal raw materials (with annual sales of about $ 40 billion).

Among the many achievements of genetic engineering that have been used in medicine, the most significant is the production of human insulin on an industrial scale.

Currently, according to WHO, there are about 110 million people suffering from diabetes in the world. Insulin, injections of which are indicated for patients with this disease, has long been obtained from animal organs and used in medical practice. However, long-term use of animal insulin leads to irreversible damage to many organs of the patient due to immunological reactions caused by injection of animal insulin foreign to the human body. But even the needs for animal insulin were met by only 60-70% until recently. Genetic engineers cloned the insulin gene as the first practical task. Cloned human insulin genes were injected with a plasmid into a bacterial cell, where the synthesis of a hormone that natural microbial strains have never synthesized began. Since 1982, companies in the USA, Japan, Great Britain and other countries have been producing genetically engineered insulin. In Russia, the production of genetically engineered human insulin - Insurane is carried out at the M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences. Today, domestic insulin is produced in a volume sufficient to provide diabetic patients in Moscow. At the same time, the demand of the entire Russian market for genetically engineered insulin is mainly met by imported supplies. The global insulin market is currently more than $400 million, with an annual consumption of about 2,500 kg.

The development of genetic engineering in the 80s of the last century provided a good foundation for Russia in creating genetically engineered strains of microorganisms with specified properties - producers of biologically active substances, in developing genetically engineered methods for reconstructing the genetic material of viruses, in obtaining medicinal substances, including using computer modeling. Recombinant interferon and medicinal forms based on it for medical and veterinary purposes, interleukin (b-leukin), erythropoietin have been brought to the production stage. Despite the growing demand for highly purified drugs, domestic production of immunoglobulins, albumin, plasmol provides 20% of the needs of the domestic market.

Research is actively underway to develop vaccines for the prevention and treatment of hepatitis, AIDS and a number of other diseases, as well as conjugated vaccines of a new generation against the most socially significant infections. Polymer-subunit vaccines of the new generation consist of highly purified protective antigens of various natures and a carrier – the immunostimulator polyoxidonium, which provides an increased level of a specific immune response. Russia could provide vaccinations against the vast majority of known infections on the basis of its own immunological production. Only the production of rubella vaccine is completely absent.

Genetic engineering for agriculture

Genetic improvement of agricultural crops and ornamental plants is a long and continuous process using increasingly accurate and predictable technologies. The UN scientific Report (for 1989) says the following: "Because molecular methods are the most accurate, those who use them are more confident in what traits they endow plants with, and therefore less likely to get unplanned effects than when using conventional breeding methods."

The advantages of new technologies are already widely used in countries such as the USA, Argentina, India, China and Brazil, where genetically modified crops are cultivated over large areas.

New technologies are also of great importance for poor farmers and residents of poor countries, especially women and children. For example, genetically modified, pest-resistant cotton and corn require the use of insecticides in much smaller volumes (which makes farm work safer). Such crops contribute to higher yields, higher incomes for farmers, lower poverty and the risk of poisoning the population with chemical pesticides, which is especially typical for a number of countries, including India, China, South Africa and the Philippines.

The most common GM plants are crops resistant to inexpensive, least toxic and most widely used herbicides. The cultivation of such crops allows you to get a higher yield per hectare, get rid of exhausting manual weeding, spend less money due to minimal or ploughless cultivation of the land, which, in turn, leads to a decrease in soil erosion.

In 2009, genetically modified crops of the first generation were replaced with second-generation products, which for the first time led to an increase in yield per se. An example of a new class of biotechnological culture (which many researchers have been working on) is the glyphosate–resistant soybean RReady2Yield™ , which was grown in 2009 in the USA and Canada on more than 0.5 million hectares.

The introduction of genetic engineering into modern agrobiology can be illustrated by the following facts from a number of foreign expert reviews, including the annual review of the independent International Service for Monitoring the Use of Agrobiotechnologies (ISAA), headed by the world-famous expert Clive James (Claiv James): (www.isaaa.org )

In 2009, GM crops were grown in 25 countries on an area of 134 million hectares (which is 9% of 1.5 billion hectares). ha of all arable land in the world). Six EU countries (out of 27) cultivated Bt corn, and in 2009 the area of its crops reached more than 94,750 hectares. An analysis of the global economic effect of the use of biotechnological crops for the period from 1996 to 2008 shows an increase in profit of $ 51.9 billion due to two sources: firstly, it is a reduction in production costs (50%) and, secondly, a significant increase in yield (50%) in the amount of 167 million tons.

In 2009, the total market value of GM seeds in the world amounted to 10.5 billion dollars. The total value of biotech corn and soybeans, as well as cotton, in 2008 amounted to $ 130 billion, and it is expected that its annual growth will be 10-15%.

It is estimated that in case of full adoption of biotechnology, by the end of the period 2006 – 2015, the profit of all countries in terms of GDP will grow by 210 billion US dollars per year.

Observations carried out since the beginning of the use of herbicide-resistant crops in agriculture convincingly prove that farmers have been able to fight weeds more effectively. At the same time, loosening and plowing fields lose their importance as a means of weed control. As a result, tractor fuel consumption is reduced, the soil structure is improved and its erosion is prevented. Targeted insecticidal programs for Bt cotton cultivation provide for fewer spraying of crops and, consequently, fewer visits of equipment to the fields, which leads to a reduction in soil erosion. All this unwittingly contributes to the introduction of preservative tillage technology aimed at reducing soil erosion, carbon dioxide levels and reducing water loss.

The current state of science is characterized by an integrated approach, the creation of unified technological platforms for a wide range of research. They combine not only biotechnology, molecular biology and genetic engineering, but also chemistry, physics, bioinformatics, transcriptomics, proteomics, and metabolomics.

Recommended literature
1. J. Watson. Molecular biology of the gene. M.: Mir. 1978.
2. Stent G., Kalendar R. Molecular genetics. M.: Mir. 1981
3. S.N. Shchelkunov "Genetic engineering". Novosibirsk, Siberian University Press, 2008
4. Glick B. Molecular biotechnology. Principles and application / B. Glick, J. Pasternak. M.: Mir, 2002
5. Genetic engineering of plants. Laboratory manual. Edited by J. Draper, R. Scott, F. Armitage, R. Walden. M.: "The World". 1991.
6. Agrobiotechnology in the world. Ed. Scriabina K.G. M.: Bioengineering Center of the Russian Academy of Sciences, 2008– - 135 p.
7. Clark. D., Russell L. Molecular biology is a simple and entertaining approach. Moscow: CJSC "Company COND". 2004

1. "On state regulation of genetic engineering activities". FZ-86 as amended in 2000, Article 1
2. The Cologne Protocol, Cologne Paper, was adopted at the conference "Towards a Knowledge-based Bioeconomy" (Cologne, May 30, 2007), organized by the European Union during the German presidency of the EU.
3. http://www.www.essentialbiosafety.info

Source: Encyclopedia of the Lomonosov Knowledge Foundation.

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