6.1 Genetic engineering, principles, possibilities. Areas of application of biological agents obtained by genetic engineering methods

When optimizing any biotechnological process involving living organisms, the main efforts are usually aimed at improving their genetic properties. Traditionally, mutagenesis was used for these purposes, followed by screening and selection of suitable variants. Today, enormous changes have taken place in this area. Currently, fundamentally new methods based on recombinant DNA technology are being developed and applied. Modification of genetic material is carried out by different methods: in a living organism (in vivo) and outside it (in vitro), respectively, these are two directions - cellular engineering

riya and genetic engineering.

Using these methods, it is possible to obtain new highly productive producers of human proteins and peptides, antigens, viruses, etc. The development of genetic and cellular engineering leads to the fact that the biotechnological industry is increasingly conquering new areas of production. The foundation for the emergence of the latest methods of biotechnology were discoveries in genetics, molecular biology, genetic enzymology, virology, microbiology and other disciplines.

The rapid introduction of the latest fundamental advances into practice and the significant influence of the latter on the level of theoretical research inherent in biotechnology are most clearly demonstrated by the development of genetic engineering.

The most important stage in the development of biotechnology was the separation of molecular biology into an independent discipline in the middle of the current century. The emergence of molecular biology became possible thanks to the interaction of genetics, physics, chemistry, biology, mathematics, etc. E. Chargoff and Z. D. Hotchkiss, studying the molecular relationships of nucleotide bases in DNA (adenine, guanine, cytosine, thymine) showed that in various organisms they are the same. This discovery played a key role in establishing the structure of DNA. Progress in the field of genetics of bacteria and bacteriophages played a major role in deciphering the structure of DNA. It was established (A. Hershey, M. Chase, J. Lederberg, N. Zinder) that transduct-

tion (transfer of genetic material) can be carried out with the help of a bacteriophage, and phage DNA can play the role of a carrier of heredity. B. Hayes also clarified the patterns of the sexual process in bacteria(conjugation) , in which from donor cells having F-factor (fertility), genetic material is transferred to the recipient

ent cells. J. Watson and F. Crick proposed a complementary model of the structure of DNA and the mechanism of its replication; A unique property of DNA was discovered - the ability of self-reproduction (replication).

Based on molecular biology and genetics of microorganisms, by the early 60s. molecular genetics was formed. G. Gamow in 1954 put forward the hypothesis that each codon (a sequence of nucleotides encoding one amino acid) should consist of three nucleotides. In 1961, it was confirmed experimentally that the primary structure of a protein is encoded in DNA as a sequence of nucleotide triplets (codons), each of which corresponds to one of 20 amino acids. By 1966, it was possible to obtain data on the structure genetic code.

The next question was how information is transferred from DNA located in the nucleus to the cytoplasm, where protein synthesis occurs on ribosomes. It was found that the sequence of triplet codons stored in DNA transcribed(rewritten) into short-lived molecules of in-

formational RNA (mRNA). This DNA → mRNA stage was called trans-

transcription, and the mRNA → protein stage – translation . The transfer of an amino acid and the determination of its location in the synthesized protein molecule is carried out by transfer RNA (tRNA) . RNA is synthesized on DNA, as a matrix, and protein is synthesized on RNA. Some viruses lack the first link, and RNA serves as the material of heredity for them.

Mechanism of control of gene activity for a long time remained unknown. Great value had the work of F. Jacob and J. Monod, which showed that bacteria have structural genes, providing information about the synthesis of certain proteins and regulatory genes which turn on or off individual genes or their blocks. It further turned out that gene regulation according to this principle also occurs in other organisms. There are also other regulatory mechanisms.

The next important step was to carry out work on deciphering the nucleotide sequences (sequencing), which provides information about the primary structure of a genome region that performs certain functions. Structure and functions have acquired a general molecular biological expression; its essence lies in the fact that functional states express structural changes in macromolecules and associations.

From studying the patterns of functioning of genetic material in a cell, researchers soon moved on to genetic manipulation. A new experimental technology has emerged that involves introducing foreign genes into cells. The names “genetic (or genetic) engineering” or “working with recombinant DNA” are equivalent. The essence of this techno-

logic is in the reunification of DNA fragments in vitro with subsequent

general introduction of new (“recombinant”) genetic structures into a living cell.

In 1972, Berg and his colleagues created the first recombinant DNA molecule, consisting of a DNA fragment of the OB40 virus and a bacteriophage

6. CELLULAR AND GENETIC ENGINEERING

6.1 Genetic engineering, principles, possibilities. Application areas biological. agents obtained by genetic methods. engineering

λ dvgal with galactose operon E. coli. Two groups of enzymes became tools for genetic design: restriction endonucleases (restriction enzymes) and ligases. The first are necessary to obtain one

native DNA fragments, the second - for their connection. Restriction enzymes and ligases, together with other enzymes (nucleases, reverse transcriptase, DNA polymerase, etc.) ensure all genetic engineering manipulations.

The E. coli plasmid is cleaved by a restriction enzyme in both parts of the DNA to form unpaired nucleotides (TTAA or AATT) at the ends. The gene is cleaved using the same restriction enzyme with the formation of sequences (AATT and TTAA) at the ends that are complementary to the plasmid. Both DNA (gene and plasmid) are fused using ligase. The hybrid plasmid is introduced into E. coli, which upon reproduction forms a clone, all cells of which contain the recombinant plasmid and the foreign gene. The gene is cloned in a bacterial cell and induces protein synthesis in it.

The in vitro genetic engineering technique includes several sequential procedures (slide):

- obtaining the desired gene;

- its integration into a genetic element capable of replication (vector);

- introduction of the gene included in the vector into recipient organism;

- identification (screening) and selection of cells that have acquired the desired gene or genes.

Obtaining genes. Genes can be obtained in several ways: isolation from DNA, chemical-enzymatic synthesis and enzymatic synthesis.

Isolating genes from DNA carried out using restriction enzymes that catalyze the cleavage of DNA in areas that have certain nucleotide sequences (4–7 nucleotide pairs). Cleavage can be carried out in the middle of a recognizable region of nucleotide pairs; in this case, both DNA strands are “cut” at the same level. The resulting DNA fragments have so-called blunt ends. DNA cleavage is possible with a shift, with one of the strands protruding by several nucleotides. The “sticky” ends formed in this case, due to their complementarity, interact.

A nucleotide sequence with sticky ends can be attached to a vector (pre-treated with the same restriction enzyme) and converted into a circular sequence as a result of cross-linking of mutually complementary ends with ligases. The method has significant drawbacks, since it is quite difficult to select the action of enzymes for strict isolation of the desired gene. Along with the gene, “extra” nucleotides are captured or, conversely, enzymes cut off part of the gene, turning it into a functionally defective one.

Chemical-enzymatic synthesis is used if the primary structure of the protein or peptide whose synthesis the gene encodes is known. Full knowledge of the nucleotide sequence of the gene is necessary. This method allows

6. CELLULAR AND GENETIC ENGINEERING

6.1 Genetic engineering, principles, possibilities. Application areas biological. agents obtained by genetic methods. engineering

accurately recreate the desired nucleotide sequence, and also enter into

genes, recognition sites for restriction enzymes, regulatory sequences, etc. The method consists of the chemical synthesis of single-stranded DNA fragments (oligonucleotides) due to the step-by-step formation of ester bonds between nucleotides, usually 8–16 units. Currently, there are “gene machines” that, under the control of a microprocessor, very quickly synthesize specific short sequences of single-stranded DNA. The slide shows a diagram of such a machine, designed by the Canadian company Bio Logics. The desired sequence of bases is entered onto the keypad. The microprocessor opens valves through which nucleotides, as well as the necessary reagents and solvents, are sequentially supplied to the synthesis column using a pump. The column is filled with silicon beads on which DNA molecules are collected. This device can synthesize chains up to 40 nucleotides in length at a rate of 1 nucleotide in 30 minutes. The resulting oligonucleotides are cross-linked with each other using DNA ligase to form a double-stranded nucleotide. Using this method, genes for the A- and B-chains of insulin, proinsulin, somatostatin, etc. were obtained.

Enzymatic gene synthesis based on isolated messenger RNA

(mRNA) is currently the most common method. First, messenger RNAs are isolated from cells, among which there is mRNA encoded by the gene that needs to be isolated. Then, under selected conditions, on the mRNA isolated from the cell, as on a matrix, using reverse transcriptase (revertase) a DNA strand complementary to the mRNA (cDNA) is synthesized. Received complementary DNA(cDNA) serves as a template for the synthesis of the second strand of DNA using DNA polymerase or reversease. The primer in this case is an oligonucleotide complementary to the 3’ end of the mRNA; a new DNA strand is formed from deoxynucleoside triphosphates in the presence of magnesium ions. The method was used with great success to obtain the gene for human growth hormone (somatotropin) in 1979.

The gene obtained in one way or another contains information about the structure of the protein, but cannot implement it itself. Therefore, additional mechanisms are needed to control the action of the gene.

The transfer of genetic information into the recipient cell is carried out as part of a vector. A vector is, as a rule, a circular DNA molecule capable of independent replication. The gene together with the vector forms recombinant DNA.

Construction of recombinant DNA. With normal administration

bacterial cell DNA is subjected to enzymatic attack, as a result of which it is destroyed. To prevent this from happening, vector DNA molecules are used that, when introduced into a cell, can exist autonomously and replicate when the cell divides. The vector also contains a genetic trait necessary for subsequent recognition and selection of transgenic organisms. Antibiotic resistance genes are usually used as marker genes.

6. CELLULAR AND GENETIC ENGINEERING

6.1 Genetic engineering, principles, possibilities. Application areas biological. agents obtained by genetic methods. engineering

The construction of recombinant DNA is carried out in vitro with isolated DNA using restriction endonucleases, which cleave the vector in one site, converting it from a circular form to a linear one with the formation of sticky ends complementary to the ends of the input DNA. The complementary ends of the vector and the introduced gene are joined by ligase. The resulting recombinant DNA is closed using the same DNA ligase to form a circular molecule.

Plasmids and viruses are used as vectors. Viruses are transported from cell to cell and can quickly infect the entire body in a short time. An important problem when using them is attenuation (weakening of pathogenicity for the host); therefore, it is not obvious that cells infected with the virus will survive and be able to pass on the altered genetic program to their offspring. The most common vectors are multicopy plasmids with a molecular weight of 3–10 kb. The first plasmids were isolated from bacteria, and subsequently they began to be constructed using genetic engineering methods.

Using general-purpose vectors methodically is a simple task that does not require special equipment. The most used plasmid vectors for cloning are E. coli plasmids (pBR322, pBR325, pACYC117, pACYC 184), as well as those constructed on the basis of the CoIEI plasmid. Modern plasmid vectors in the presence of chloramphenicol are capable of replication, regardless of chromosome division; the number of plasmid copies can increase to 1–2.103 copies per cell.

When obtaining a library of genes from plants and higher animals, in which the total genome length is up to 3109 or more, the capacity of the vector often plays a decisive role. In this case, phage λ DNA is used as a vector. Using special methods, recombinant DNA is introduced directly into the phage heads. Cosmid plasmids have an even greater capacity (up to 40 kb), in which the cos fragment of the λ phage genome is involved in DNA packaging into the phage particle at the final stage of development. For DNA packaging, the DNA must contain a COS region and be approximately the same size as the phage genome. The achieved methods of packaging DNA into a phage head using cosmids make it possible to obtain gene libraries from almost any organism.

Transfer of genes into cells of the recipient organism. Transfer of recombinant

nant DNA is carried out by transformation or conjugation. Transformation is the process of changing the genetic properties of a cell as a result of the penetration of foreign DNA into it. It was first discovered in pneumococci by F. Giffith, who showed that some cells of non-virulent strains of bacteria, when they infect mice together with virulent strains, acquire pathogenic properties. In the future

6. CELLULAR AND GENETIC ENGINEERING

6.1 Genetic engineering, principles, possibilities. Application areas biological. agents obtained by genetic methods. engineering

transformation has been demonstrated and studied in various bacterial species.

It has been established that only a few, so-called competent cells (capable of incorporating foreign DNA and synthesizing a special transforming protein) are capable of transformation. The competence of a cell is also determined by environmental factors. This can be facilitated by treating cells with polyethylene glycol or calcium chloride. After penetration into the cell, one of the recombinant DNA strands degrades, and the other, due to recombination with a homologous region of the recipient DNA, can be included in a chromosome or extrachromosomal unit. Transformation is the most universal way of transmitting genetic information and has highest value for genetic technologies.

Conjugation is one of the methods of exchange of genetic material, in which a unidirectional transfer of genetic information occurs from the donor to the recipient. This transfer is under the control of special conjugative plasmids (fertility factor). The transfer of information from the donor cell to the recipient cell is carried out through special genital villi (pili). It is also possible to transfer information using non-conjugative plasmids with the participation of helper plasmids.

The transfer of the entire set of genes of a virus or phage, leading to the development of phage particles in the cell, is called transfection. The technique, as applied to bacterial cells, involves obtaining spheroplasts, purifying the incubation medium from nucleases, and adding purified phage DNA (the presence of protamine sulfate increases the efficiency of transfection). The technique is applicable to animal and plant cells using special shuttle viral vectors.

Screening and selection of recombinant cells. After transferring the design

ruated DNA, as a rule, only a small part of the recipient cells acquires the necessary gene. Therefore, a very important step is the identification of cells carrying the target gene.

At the first stage, cells carrying the vector on the basis of which DNA transfer is carried out are identified and selected. Selection is carried out using genetic markers that mark the vector. The main markers are antibiotic resistance genes. Therefore, selection is carried out by seeding cells on media containing a specific antibiotic. After seeding on these media, only cells that contain a vector with antibiotic resistance genes grow.

At the second stage, cells carrying the vector and the target gene are selected. For this, two groups of methods are used: 1) based on direct analysis of the DNA of recipient cells and 2) based on the identification of the trait encoded by the target gene. When using the first group of methods, vector DNA is isolated from cells supposedly containing the desired gene, and a search is carried out for regions carrying this gene. Next, part of the nucleotide sequence of the gene is sequenced.

6. CELLULAR AND GENETIC ENGINEERING

6.1 Genetic engineering, principles, possibilities. Application areas biological. agents obtained by genetic methods. engineering

Another method is possible - hybridization of DNA isolated from cells with a probe (the desired gene or its corresponding mRNA); The isolated DNA is converted into a single-stranded state and interacts with the probe. Next, the presence of double-stranded hybrid DNA molecules is determined. In the second option, it is possible to directly select cells that synthesize protein - the product of transcription and translation of the target gene. Selective media are also used that support the growth of only cells that have acquired the gene target.

Using genetic engineering methods, it is possible to construct new forms of microorganisms according to a given plan, capable of synthesizing a variety of products, including eukaryotic organisms. Recombinant microbial cells quickly multiply under controlled conditions and are able to utilize a variety of substrates, including inexpensive ones.

The main problems that arise during genetic manipulation are as follows: 1) during transformation, genes entering a foreign environment are exposed to proteases, so they must be protected; 2) as a rule, the product of the transplanted gene accumulates in cells and is not released into the environment; 3) most desirable traits are encoded not by one, but by a group of genes. All this significantly complicates transfer and requires the development of technology for sequential transplantation of each gene.

To date, genetic engineering has mastered all living kingdoms. The phenotypic expression of “foreign” genes (expression) has been obtained in bacteria, yeast, fungi, plants and animals. Brilliant successes have been achieved on the cells of the most comprehensively studied microorganisms. The era of recombinant DNA in plants and higher animals is just beginning. In the field of genetic engineering of animals, cloning

genes of mouse β-globin, phage λ. In addition to African green monkey kidney cells, new types of animal cell culture are being tested, including human cells. For example, in gypsy moth cells using a viral vector, it was possible to achieve expression of the human β-interferon gene. This gene has also been cloned in mammalian cells. In human genetic engineering, as in the genetic engineering of plants, tissue-specific gene expression has not yet been achieved. Solutions to this problem are sought by introducing certain promoter regulatory regions into constructed vectors. The possibility of improving agricultural breeds of animals remains a rather distant task. To date, there is practically no information on the genetics of such traits as fertility, milk yield and fat content, increased resistance to diseases, etc. This hinders attempts at genetic manipulation in this area.

Genetic engineering not only puts new producers of valuable compounds into the hands of biotechnologists, but also improves and increases the efficiency of valuable properties of traditionally used organisms. Common

6. CELLULAR AND GENETIC ENGINEERING

6.1 Genetic engineering, principles, possibilities. Application areas biological. agents obtained by genetic methods. engineering

One of the best methods for increasing the yield of useful product is amplification

tion – increase in the number of gene copies . Education of many target pro-

products (amino acids, vitamins, antibiotics, etc.) is characterized by a long biochemical synthesis pathway, which is controlled not by one, but by dozens of genes. Isolation of these genes and cloning using amplification is a rather difficult, but in some cases possible, task. An increase in the yield of useful product is also achieved using local

lysed (site-specific) mutagenesis in vitro : using chemical mutagenesis, not the entire genome of the cell is processed, but its fragment obtained through restriction.

6.2 Technologies for genetic design of organisms in vitro. Sources of DNA for gene cloning (restriction, enzymatic and chemical-enzymatic gene synthesis).

Methods for introducing DNA. Expression of genes in recombinant DNA. Genetic engineering of industrially important producers of insulin, somatotropin, interferons

The development of recombinant DNA technology makes it possible to isolate eukaryotic genes and express them in heterologous systems. Currently, genetic engineering methods make it possible to construct genetic systems capable of functioning in prokaryotic and eukaryotic cells. These capabilities are used to create organisms with new valuable properties, for example, bacterial strains capable of synthesizing eukaryotic proteins.

Among the protein products of great interest are biologically active substances such as hormones. Protein and peptide hormones occupy an important place among them. These hormones, many of which are urgently needed in medicine, until recently were obtained by extraction from animal tissues, provided that the hormone did not have a pronounced species specificity. Attempts were made to obtain relatively short peptide hormones by chemical synthesis. But this method of production turned out to be unprofitable even for molecules consisting of several dozen units. The only source of hormones with extremely pronounced species specificity (growth hormone somatotropin) were the organs of deceased people.

Advances in genetic engineering have raised hopes for the possibility of cloning genes for the synthesis of a number of hormones in microbial cells. These hopes were largely justified, first of all, by the example of the microbiological synthesis of peptide hormones.

The first successful results on the expression of a chemically synthesized DNA nucleotide sequence encoding the 14-mer peptide hormone somatostatin (somatotropin antagonist) were obtained in 1977 in the USA by the Genetek company. To prevent the destruction process

6. CELLULAR AND GENETIC ENGINEERING

To study the hormone in bacterial cells under the influence of peptidase, the authors used an approach that was later successfully used to obtain other peptide hormones. A hybrid gene was constructed, part of which was taken from the gene for the β-galactosidase enzyme of Escherichia coli, and the remainder was a fragment encoding somatostatin itself (the fragment was synthesized chemically). Entered in bacterial cells the hybrid gene directed the synthesis of a chimera protein consisting of more than 90% of the amino acid sequence of β-galactosidase. The rest was somatostatin. At the junction of the region of the two original genes there was a codon for the amino acid methionine. The latter made it possible to treat the hybrid protein with cyanogen bromide, which breaks the peptide bond formed by methionine; Somatostatin was found among the breakdown products. This approach has been used to obtain many peptide hormones (A- and B-chains of insulin, neuropeptide lehenkephalin, bradykinin, angiotensin, etc.).

Using genetic engineering methods, super-producing microorganisms have been created in a short period of time, making it possible to obtain a number of viral and animal proteins in high yields. Strains have been created in which up to 20% of the cellular protein consists of genetically engineered products, for example, cow antigen of the hepatitis B virus, the main capsid antigen of the foot-and-mouth disease virus, calf rennin, surface antigen of the hepatitis B virus, etc.

Preparation of recombinant insulin. The hormone insulin is built from two polypeptide chains, A and B, 20 and 30 amino acids long, respectively. The sequence of circuits was established in 1955 by Sanger. The synthesis of both chains, including 170 chemical reactions, was realized in the USA, Germany and China in 1963. But it turned out to be impossible to transfer such a complex process to industry. Until 1980, insulin was obtained by isolating it from the pancreas (the pancreas of a cow weighs 200–250 g, and to obtain 100 g of crystalline insulin requires up to 1 kg of raw materials). Therefore, the needs for it were not fully satisfied. Thus, in 1979, out of 6 million registered patients with diabetes, only 4 million people received insulin. In 1980, the Danish company Novo Industry developed a method for converting pig insulin into human insulin by enzymatic replacement of the alanine residue, which is 30th amino acid in chain B, to the threonine residue. As a result, single-component human insulin 99 was obtained% cleanliness. In the animal's body, two polypeptide chains are initially parts of one protein molecule 109 amino acids long - this is preproinsulin. When synthesized in pancreatic cells, the first 23 amino acids serve as a signal for the transport of the molecule across the cell membrane. These amino acids are split off to form proinsulin, which is 86 amino acids long.

In 1980, Gilbert and colleagues isolated insulin mRNA from a rat pancreatic β-cell tumor (manipulation was not allowed at that time).

6. CELLULAR AND GENETIC ENGINEERING

6.2 Technologies for genetic design of organisms nvitro. Sources of DNA for gene cloning. DNA injection methods...

to be influenced by human genes). The resulting DNA copy of the mRNA was inserted into the pBR 322 plasmid, into middle part penicillinase gene (the enzyme is normally released from the cell), which was transported into the bacterium. The constructed plasmid turned out to contain information about the structure of proinsulin, and not preproinsulin. During translation of mRNA in E. coli cells, a hybrid protein containing penicillinase and proinsulin sequences was synthesized. The hormone from this protein was cleaved with trypsin. It has been proven that the protein obtained in this way affects sugar metabolism in a similar way to the pancreatic hormone. In 1979, in the United States, genes encoding the A- and B-chains of insulin were synthesized within three months; genes were assembled from 18 and 11 oligonucleotides, respectively. Next, the genes were inserted, as in the production of somatostatin, into a plasmid at the end of the β-galactosidase gene of Escherichia coli.

In E. coli cells, proinsulin is also synthesized, and not just its individual chains. A DNA copy was synthesized from the isolated template mRNA. Synthesis of proinsulin has certain advantages, since the procedures for extraction and purification of the hormone are minimal.

Improving the technology for obtaining genetically engineered producer strains using various techniques (plasmid amplification, encapsulation of introduced recombinant DNA, suppression of the proteolytic activity of recipient cells) made it possible to obtain high yields of the hormone, up to 200 mg/l of culture. Medical, biological and clinical tests of the genetically engineered protein showed the suitability of the drug, and in 1982 it was approved for production in many countries.

Biosynthesis of somatotropin. Somatotropin (pituitary growth hormone) was first isolated in 1963 from cadaveric material. The hormone output from one pituitary gland was about 4–6 mg in terms of the finished pharmaceutical preparation. For the treatment of dwarfism, the required dose is 6 mg per week for a year. In addition to the lack of mass, the drug obtained by extraction was heterogeneous; antibodies were produced against it, which negated the effect of the hormone. Moreover, there was a danger that when receiving the drug, the body could become infected with slowly developing viruses. Therefore, children receiving this drug required many years of medical supervision.

The genetically engineered drug has undoubted advantages: it is available in large quantities, homogeneous, does not contain viruses. The synthesis of somatotropin, consisting of 191 amino acid residues, was carried out in the USA by Goeddel and co-workers in 1979 (Genentek company).

Chemical-enzymatic DNA synthesis produces a gene encoding a somatotropin precursor, so a special cloning route was chosen. At the first stage, a double-stranded DNA copy of the mRNA was cloned and, by restriction digestion, a sequence encoding the entire amino acid sequence of the hormone was obtained, except for the first 23 amino acids. Next, a synthetic polynucleotide corresponding to these 23 amino acids with the ANG start codon at the beginning was cloned. Two received-

6. CELLULAR AND GENETIC ENGINEERING

6.2 Technologies for genetic design of organisms nvitro. Sources of DNA for gene cloning. DNA injection methods...

These fragments were connected and adjusted to a pair of lac promoters and a ribosome binding site. The engineered gene was transplanted into E. coli. The hormone synthesized in bacteria had the required molecular weight and was not associated with any protein; its yield was about 100,000 molecules per cell. The hormone, however, contained an additional methionine residue at the N-terminus of the polypeptide chain; when the latter was removed, the hormone yield was low.

In 1980, evidence was obtained that genetically engineered somatotropin has the biological activity of the native hormone. Clinical trials of the drug were also successful. In 1982, the hormone was also obtained on the basis of engineered Escherichia coli at the Pasteur Institute in Paris. By 1990, the cost of the hormone had dropped to $5/unit. Currently, it is being used in livestock farming to stimulate the growth of livestock, milk yield, etc.

Obtaining interferons. Interferons are a group of proteins that can be produced in the nuclear cells of vertebrates. These are powerful inducible proteins that are a factor of nonspecific resistance that maintains the homeostasis of the body. The interferon system has a regulatory function in the body, as it is capable of modifying various biochemical processes. Vertebrate interferons, including

including humans, are divided into three groups: α, β, γ, respectively, leukocyte, fibroblast and immune.

In the late 70s, the potential importance of interferons for medicine, including the prevention of cancer, became obvious. Clinical trials have been hampered by the lack of sufficient quantities of interferons and the high cost of drugs obtained in the traditional way (isolation from the blood). So, in 1978, an ode to receive

0.1 g of pure interferon in the Central Health Laboratory of Helsinki (the laboratory is the world leader in the production of interferon from leukocytes healthy people) were obtained by processing 50,000 liters of blood. The resulting amount of the drug could provide treatment against viral infection in 10,000 cases. The prospects for obtaining interferons were associated with genetic engineering.

IN In 1980, Gilbert and Weissman in the USA succeeded in obtaining interferon in genetically engineered E. coli. The initial difficulty they encountered was low levels of mRNA in white blood cells, even those stimulated by virus infection. By processing 17 liters of blood, it was possible to isolate mRNA and obtain DNA copy. The latter was inserted into a plasmid and cloned into E. coli. Over 20,000 clones were tested. Some clones were capable of synthesizing interferon, but with low yield, 1–2 molecules per cell. Similar studies were carried out in Japan, England, France, and Russia.

IN 1980 nucleotide sequences were establishedα - and β - interferons: fibroblast interferon mRNA consists of 836 nucleotides

6. CELLULAR AND GENETIC ENGINEERING

6.2 Technologies for genetic design of organisms nvitro. Sources of DNA for gene cloning. DNA injection methods...

Dov; of these, 72 and 203 nucleotides are in the 5’ and 3’ untranslated regions, 63 encode the peptide responsible for the secretion of interferon from cells, and 498 nucleotides encode 166 amino acid residues of interferon itself. After this, the α- and β-interferon genes were obtained by chemical synthesis and cloned into E. coli. In 1981, the nucleotide sequence of immune interferon was deciphered, significantly different from the first two, but comparable in size of the molecule. A significant point was the complete synthesis of the human leukocyte interferon gene, carried out in the UK by employees of the Imperial Chemical Industry company and the School of Biological Sciences at the University of Leicester. Within a year and a half, the complete sequence of a DNA copy of interferon was synthesized, capable of encoding α 1-interferon. The synthesis of oligonucleotides was carried out by a new method, which significantly accelerated gene synthesis. First, a nucleotide was attached to the polyacrylamide resin; Next, the addition of nucleotide pairs was carried out using a condensing agent in anhydrous pyridine. Each cycle lasted an hour and a half, so within a year it was possible to synthesize a sequence 5000 nucleotides long. 67 oligonucleotides were synthesized and combined using ligase into double-stranded DNA consisting of 514 nucleotide pairs. The resulting gene was inserted into the cells of two bacteria: E. coli,

Methylophilus methylotrophus, and expression was obtained.

Efforts aimed at obtaining genetically engineered interferons, compared with the cell culture method, have reduced costs by more than 100 times. Various types of interferons have been obtained based on genetically engineered bacterial and yeast cells. This made it possible to launch biomedical and clinical trials of drugs. The interferon preparations obtained during 1980–1981 were 80% purified and had a specific activity of more than 107 international units per 1 mg of protein. The expansion of clinical trials of interferons begun during this period depends on an increase in the degree of its purification. Progress in this direction has been achieved with the use of monoclonal antibodies, which can be used for affinity chromatography (in which the desired proteins are retained on a column with antibodies).

6.3 Cellular engineering. Production of biological agents using cell engineering methods in vivo. Mutagenesis.

Methods for obtaining and isolating mutants. Hybridization of eukaryotic cells.

Plasmids and conjugation of bacteria. Phagic transduction. Technique of protoplast fusion. Hybridomas.

Preparation and use of monoclonal antibodies

6. CELLULAR AND GENETIC ENGINEERING

Traditionally, to obtain more active biological agents,

changed selection and mutagenesis. Selection is the directed selection of mutants

– organisms whose heredity has acquired an abrupt change as a result of a structural modification in the nucleotide sequence of DNA. The general path of selection is the path from the blind selection of the desired producers to the conscious design of their genome. Traditional selection methods once played an important role in the development of various technologies using microorganisms. Strains of beer, wine, baking, acetic acid and other microorganisms were selected. The limitations of the selection method are associated with the low frequency of spontaneous mutations leading to changes in the genome. A gene must double on average 106–108 times for a mutation to occur.

Leads to a significant acceleration of the selection process induced mutagenesis(a sharp increase in the frequency of mutations of a biological object due to artificial damage to the genome). Ultraviolet and X-ray radiation and a number of chemical compounds (nitrous acid, bromuracil, antibiotics, etc.) have a mutagenic effect. After treating the population with a mutagen, a total screening (verification) of the resulting clones is carried out and the most productive ones are selected. The selected clones are reprocessed and productive clones are again selected, that is, stepwise selection is carried out according to the trait of interest.

This work requires a lot of labor and time. The disadvantages of stepwise selection can be largely overcome by combining it with genetic exchange methods.

Genetic design in vivo (cell engineering) includes obtaining and isolating mutants and using in various ways exchange of hereditary information of living cells

The basis of cell engineering is the fusion of non-reproductive cells (hybridization of somatic cells) to form a single whole. Cell fusion can be complete, or the recipient cell can acquire individual parts of the donor cell (mitochondria, cytoplasm, nuclear genome, chloroplasts, etc.). Recombination is caused by various processes of exchange of genetic information of living cells (sexual and parasexual processes in eukaryotic cells; conjugation, transformation and transduction in prokaryotes, as well as the universal method - protoplast fusion).

During hybridization, genetically marked strains of microorganisms are taken (usually auxotrophic mutants or mutants resistant to growth inhibitors). As a result of cell fusion (copulation), hybrids are formed in yeast, fungi, and algae. If the original cells were haploid, as a result of nuclear fusion, a diploid cell (zygote) appears, carrying a double set of chromosomes in the nucleus. In some representatives, the nucleus immediately undergoes meiosis, during which each of the chromosomes is split. Homologous chromosomes form pairs and exchange parts of their chromatids as a result of crossing over. Next, haploid sexual spores are formed, each of which contains a set of genes that distinguished the parent cells as a result of gene recombination

6. CELLULAR AND GENETIC ENGINEERING

6.3 Cellular engineering. Receive biological agents using cell methods. in vivo engineering. Mutagenesis. Receiving methods and isolation of mutants

the same chromosome, as well as different chromosomes in the distribution of chromosome pairs. If after fusion the nuclei do not fuse, forms with mixed cytoplasm and nuclei of different origins (heterokaryons) are formed. Such forms are characteristic of fungi, especially penicillin producers. When the resulting heterozygous diploids or heterokaryons reproduce, splitting occurs - manifestation in the offspring, which reveals not only the dominant, but also the recessive characteristics of the parents. Sexual and parasexual processes are widely used in genetic practice of industrially important producing microorganisms.

In bacteria, the exchange of genetic information occurs as a result of interaction conjugative plasmids (conjugation). For the first time

ation was observed in E. coli K-12. For conjugative crossing, the donor and recipient cultures are mixed and incubated together in nutrient broth or on the surface of agar media. The cells are connected to each other using the resulting conjugation bridge; through the bridge, a specific site of the plasmid chromosome is transferred to the recipient. Thus, at 37 °C, it takes about 90 minutes to transfer the entire chromosome. Conjugation has opened and is opening up broad prospects for genetic analysis and strain construction.

Transduction is the process of transferring genetic information from a recipient cell to a donor cell using a phage . For the first time this pro-

the process was described in 1952 by Zinder and Liederberg. Transduction is based on the fact that during the reproduction of phages in bacteria, particles can be formed that contain phage DNA and fragments of bacterial DNA. To carry out transduction, it is necessary to multiply the phage in the cells of the donor strain and then infect the recipient cells with it. The selection of recombinant forms is carried out on selective media that do not support the growth of the original forms.

IN in recent years it has been very widely usedprotoplast fusion method. This method appears to be a universal way of introducing genetic information into cells of various origins. The simplicity of the method makes it accessible for the selection of industrially important producers. The method opens up new possibilities for obtaining interspecific and intergeneric hybrids and crossing phylogenetically distant forms of living things. Positive results were obtained from the fusion of bacterial, yeast and plant cells. Interspecific and intergeneric yeast hybrids were obtained. There is evidence of the fusion of cells of various types of bacteria and fungi. It was possible to obtain hybrid cells as a result of the fusion of cells of organisms belonging to different kingdoms: animal and plant. Frog nuclear cells were fused with carrot protoplasts; a hybrid plant-animal cell grew on plant cell media, but quickly lost its nucleus and became covered with a cell wall.

IN In recent years, work has been successfully carried out to create associations of cells of different organisms, that is, mixed cultures of cells of two or more organisms are obtained in order to create artificial symbioses. Experiments on the introduction of a nitrogen-fixing organism have been successfully carried out

6. CELLULAR AND GENETIC ENGINEERING

6.3 Cellular engineering. Receive biological agents using cell methods. in vivo engineering. Mutagenesis. Receiving methods and isolation of mutants

Anabaena variabilis in tobacco plants. Attempts to introduce A. variabilis directly into cuttings of mature tobacco plants did not yield positive results. But with the joint cultivation of mesophilic tobacco tissue and cyanobacteria, it was possible to obtain regenerated plants containing cyanobacteria. Associations of ginseng and nightshade cells with cyanobacteria were obtained.

her Chlorogleae fritschii.

Clonal propagation of animal cells for genetic manipulation is promising. The technique of cell cultures of animal cells for obtaining biologically active compounds has great prospects, although it is only taking its first steps. Cultures of tumor cells or normal cells transformed in vitro retain in some cases the ability to synthesize specific products. Despite the existing difficulties, the possibility of obtaining a number of substances in animal cell culture has been shown.

An important area of ​​cell engineering is associated with the early embryonic stages. Thus, in vitro fertilization of eggs allows one to overcome infertility. With the help of hormone injections, dozens of eggs can be obtained from one animal, artificially fertilized in vitro, and implanted into the uterus of other animals. This technology is used in animal husbandry to produce monozygotic twins. A new method has been developed based on the ability of individual cells of an early embryo to develop into a normal fetus. The embryo's cells are divided into several equal parts and transplanted into recipients. This allows you to reproduce various animals in an accelerated way. Embryo manipulation is used to create embryos of various animals. The approach makes it possible to overcome the interspecies barrier and create chimeric animals. In this way, for example, sheep-goat chimeras were obtained.

The most promising direction of cell engineering is

Xia hybridoma technology. Hybrid cells (hybridomas) are formed as a result of the fusion of cells with different genetic programs, for example, normal differentiated and transformed cells. A brilliant example of the achievement of this technology are hybridomas obtained as a result of the fusion of normal lymphocytes and myeloma cells. These hybrid cells have the ability to synthesize specific antibodies, as well as unlimited growth during cultivation.

Unlike the traditional technique for producing antibodies, the hybridoma technique made it possible for the first time to obtain monoclonal antibodies (antibodies produced by the descendants of a single cell). Monoclonal antibodies are highly specific; they are directed against a single antigenic determinant. It is possible to obtain several monoclonal antibodies for different antigenic determinants, including complex macromolecules.

Monoclonal antibodies have been produced on an industrial scale relatively recently. As is known, the normal immune system is capable of producing up to a million different types of antibodies in response to foreign agents (antigens), while a malignant cell synthesizes only antibodies of one type. Myeloma cells multiply rapidly. Therefore, the culture obtained

6. CELLULAR AND GENETIC ENGINEERING

6.3 Cellular engineering. Receive biological agents using cell methods. in vivo engineering. Mutagenesis. Receiving methods and isolation of mutants

from a single myeloma cell can be maintained for a very long time. However, it is not possible to force myeloma cells to produce antibodies to a specific antigen. This problem was solved in 1975 by Caesar Milstein. Employees at the Medical Research Laboratory of Molecular Biology in Cambridge came up with the idea of ​​merging mouse myeloma cells with B lymphocytes from the spleen of a mouse immunized with a specific antigen. The hybrid cells formed as a result of fusion acquire the properties of both parent cells: immortality and the ability to secrete a huge amount of any one antibody of a certain type. These works were of great importance and opened a new era in experimental immunology.

In 1980, in the USA, Carlo M. Croce and his colleagues succeeded in creating a stable, antigen-producing, intraspecific human hybridoma by fusion of B lymphocytes from a myeloma patient with peripheral lymphocytes from a patient with subacute panencephalitis.

The main stages of obtaining hybridoma technology are as follows. Mice are immunized with the antigen, after which splenocytes are isolated from the spleen, which, in the presence of polyethylene glycol, are fused with defective tumor cells (usually defective in the enzymes of the nucleotide biosynthesis pathway - hypoxanthine or thiamine). Next, they are selected on a selective medium that allows only hybrid cells to reproduce. The nutrient medium with growing hybridomas is tested for the presence of antibodies. Positive cultures are selected and cloned. Clones are injected into animals to form a tumor that produces antibodies, or they are grown in culture. Mouse ascites fluid can contain up to 10–30 mg/ml monoclonal antibodies.

Hybridomas can be stored frozen and a dose of such a clone can be administered at any time to an animal of the same line from which the fusion cells were obtained. Currently, banks of monoclonal antibodies have been created. Antibodies are used for a variety of diagnostic and therapeutic purposes, including anticancer treatment.

An effective way to use monoclonal antibodies in therapy is to bind them to cytotoxic poisons. Antibodies conjugated with poisons track and destroy tumor cells of a certain specificity in the macroorganism.

Thus, cell engineering serves as an effective way to modify biological objects and makes it possible to obtain new valuable producers at the organ and also cellular and tissue levels.

State educational institution of higher education

vocational education

VlSU

Department of History and Religious Studies

Abstract

on the topic:

Genetic and cellular engineering. Biotechnology.

Completed by: Shipilova E.V. Gr.ZYu-110

Checked by: Associate Professor of the Department of History and

religious studies Zubkov S.A.

Vladimir 2011

1. Introduction 3

2.Possibilities of genetic engineering . Biotechnology 5

3.1. Agriculture 9

3.2 Medicine and pharmaceuticals 11

4. Cloning 14

4.1 State of therapeutic research

cloning in Russia 16

5. Problems 17

6. Conclusion 23

References 25

1. Introduction

Genetic engineering is a direction of research in molecular biology and genetics, the ultimate goal of which is to obtain, using laboratory techniques, organisms with new, including those not found in nature, combinations of hereditary properties. Genetic engineering is based on the possibility of targeted manipulation of nucleic acid fragments due to the latest advances in molecular biology and genetics. These achievements include the establishment of the universality of the genetic code, that is, the fact that in all living organisms the inclusion of the same amino acids in protein molecule encoded by the same nucleotide sequences in the DNA chain; the successes of genetic enzymology, which provided the researcher with a set of enzymes that make it possible to obtain individual genes or nucleic acid fragments in isolated form, carry out in vitro synthesis of nucleic acid fragments, and combine the resulting fragments into a single whole. Thus, changing the hereditary properties of an organism using genetic engineering comes down to constructing new genetic material from various fragments, introducing this material into the recipient organism, creating conditions for its functioning and stable inheritance.

Genetic engineering arose in the beginning. 70s 20th century Genetic engineering is based on extracting a gene (encoding the desired product) or a group of genes from the cells of an organism and combining them with special DNA molecules (so-called vectors) that can penetrate the cells of another organism (mainly microorganisms) and multiply in them , i.e. creation of recombinant DNA molecules.

Recombinant (foreign) DNA introduces new genetic and physico-biochemical properties into the recipient organism. These properties include the synthesis of amino acids and proteins, hormones, enzymes, vitamins, etc.

The use of genetic engineering methods opens up the prospect of changing a number of properties of the organism: increasing productivity, resistance to diseases, increasing growth rate, improving product quality, etc. Animals that carry a recombinant (foreign) gene in their genome are usually called transgenic, and a gene integrated into the genome recipient - transgenome. Thanks to gene transfer, new qualities arise in transgenic animals, and further selection makes it possible to consolidate them in the offspring and create transgenic lines.

Genetic engineering methods make it possible to create new plant genotypes faster than classical selection methods, and it becomes possible to purposefully change the genotype - transformation.

Genetic transformation consists mainly of the transfer of foreign or modified genes into eukaryotic cells. In plant cells, it is possible to express genes transferred not only from other plants, but also from microorganisms and even animals.

Obtaining plants with new properties from transformed cells (regeneration) is possible due to their topitotency property, i.e. the ability of individual cells in the process of realizing genetic information to develop into a whole organism.

2. Possibilities of genetic engineering. Biotechnology.

Currently, the pharmaceutical industry has gained a leading position in the world, which is reflected not only in the volume of industrial production, but also in financial means invested in this industry (according to economists, it entered the leading group in terms of the volume of purchase and sale of shares in the securities markets). An important novelty was that pharmaceutical companies included in their sphere the development of new varieties of agricultural plants and animals, and spend tens of millions of dollars a year on this, they also mobilized the production of chemicals for everyday use. Additives to construction industry products and so on. Not tens of thousands, but perhaps several hundred thousand highly qualified specialists are employed in the research and industrial sectors of the pharmaceutical industry, and it is in these areas that interest in genomic and genetic engineering research is extremely high.

Obviously, therefore, any progress in plant biotechnology will depend on the development of genetic systems and tools that will allow more efficient management of transgenes. The situation is similar to that observed in the computer industry, where in addition to increasing the volume of information processed and improving the computers themselves, we also need operating systems information management, such as Microsoft “windows”.

To cleanly excise transgenic DNA into the plant genome, homologous recombination systems borrowed from microbial genetics, such as the Cre-lox and Flp-frt systems, are increasingly being used. The future, obviously, will be controlled gene transfer from variety to variety, based on the use of pre-prepared plant material, which already contains areas of homology in the desired chromosomes necessary for the homologous insertion of the transgene. In addition to integrative expression systems, autonomously replicating vectors will be tested. Of particular interest are artificial plant chromosomes, which theoretically do not impose any restrictions on the amount of theoretical information introduced.

Scientists are searching for genes that encode new useful traits. The situation in this field is changing radically, primarily due to the existence of public databases that contain information on most genes, bacteria, yeast, humans and plants, and also due to the development of methods that allow the simultaneous analysis of the expression of large numbers of genes with very high throughput. The methods used in practice can be divided into two categories:

1. Methods that allow expression profiling: subtraction hybridization, electronic comparison of EST libraries, “gene chips”, and so on. They make it possible to establish a correlation between one or another phenotypic trait and the activity of specific genes. 2. Positional cloning consists of creating, through insertional mutagenesis, mutants with disturbances in a trait or property of interest to us, followed by cloning the corresponding gene as such, which obviously contains a known sequence (insertion). The above methods do not assume any initial information about the genes that control a particular trait. The absence of a rational component in this case is a positive circumstance, since it is not limited by our current ideas about the nature and genetic control of the specific trait that interests us.

Significant progress has been made in the practical field of creating new products for the medical industry and the treatment of human diseases

Use of genetically engineered products in medicine.

	Natural products and the scope of application of genetically engineered products
Anticoagulants	Tissue plasminogen activator (TPA) activates plasmin. An enzyme involved in the resorption of blood clots; effective in the treatment of patients with myocardial infarction.
Blood factors	Factor VIII accelerates the formation of clots; deficient in hemophiliacs. The use of genetically engineered factor VIII eliminates the risk associated with blood transfusions.
Factors stimulating colony formation	Growth factors of the immune system that stimulate the formation of white blood cells. Used to treat immunodeficiency and fight infections.
erythropoietin	Stimulates the formation of red blood cells. Used to treat anemia in patients with renal failure.
Growth factors	Stimulate differentiation and growth various types cells. Used to speed up the healing of wounds.
Human growth hormone	Used in the treatment of dwarfism.
Human insulin	Used to treat diabetes
Interferon	Prevents the proliferation of viruses. Also used to treat some forms of cancer.
Leixins	Activate and stimulate the work of various types of leukocytes. Can be used for wound healing, HIV infection, cancer,
Monoclonal- ny antibodies	The highest specificity associated with antibodies is used for diagnostic purposes. They are also used for targeted delivery of drugs, toxins, radioactive and isotope compounds to cancer tumors in cancer therapy; there are many other areas of application.
Superoxide dismutase	Prevents tissue damage by reactive hydroxy derivatives in conditions of short-term oxygen deficiency, especially during surgical operations when it is necessary to suddenly restore blood flow.
	Artificially produced vaccines (the hepatitis B vaccine was the first) are in many respects better than conventional vaccines.

Recombinant DNA technology is based on the production of highly specific DNA probes, which are used to study the expression of genes in tissues, the localization of genes on chromosomes, and identify genes with related functions (for example, in humans and chicken). DNA probes are also used in the diagnosis of various diseases.

Recombinant DNA technology has made possible an unconventional protein-gene approach called reverse genetics. In this approach, a protein is isolated from a cell, the gene for this protein is cloned, and it is modified, creating a mutant gene encoding an altered form of the protein. The resulting gene is introduced into the cell. If it is expressed, the cell carrying it and its descendants will synthesize the altered protein. In this way, defective genes can be corrected and hereditary diseases can be treated.

If the hybrid DNA is introduced into a fertilized egg, transgenic organisms can be produced that express the mutant gene and pass it on to their offspring. Genetic transformation of animals makes it possible to establish the role of individual genes and their protein products both in the regulation of the activity of other genes and in various pathological processes. With the help of genetic engineering, lines of animals resistant to viral diseases have been created, as well as breeds of animals with traits beneficial to humans. For example, microinjection of recombinant DNA containing the bovine somatotropin gene into a rabbit zygote made it possible to obtain a transgenic animal with hyperproduction of this hormone. The resulting animals had pronounced acromegaly.

3. Directions of genetic engineering.

3. 1 Agriculture.

Genetic engineering directly in agriculture took place already at the end of the 1980s, when it was possible to successfully introduce new genes into dozens of species of plants and animals - to create tobacco plants with luminous leaves, tomatoes that easily tolerate frost, and corn that is resistant to pesticides.

One of the important tasks of genetic engineering is to obtain plants that are resistant to viruses, since currently there are no other ways to combat viral infections of crops. The introduction of virus envelope protein genes into plant cells makes plants resistant to this virus. Currently, transgenic plants have been obtained that can resist the effects of more than a dozen different viral infections.

Another important task of genetic engineering is related to protecting plants from insect pests. The use of insecticides is not always effective due to their toxicity and the possibility of insecticides being washed off from plants by rainwater. In genetic engineering laboratories in Belgium and the USA, work has been successfully carried out to introduce genes from the earth bacterium Bacillus thuringiensis into plant cells, which make it possible to synthesize insecticides of bacterial origin. These genes were introduced into the cells of potatoes, tomatoes and cotton, as a result of which transgenic potato and tomato plants became resistant to the Colorado potato beetle, and cotton plants turned out to be resistant to various insects, including the cotton bollworm. The use of genetic engineering in agriculture has reduced the use of insecticides by 40 - 60%. Genetic engineers have bred transgenic plants with an extended period of fruit ripening. This makes it possible to pick such tomatoes from the bush red with confidence that they will not overripe during transportation.

The list of plants to which genetic engineering methods have been successfully applied is growing. It includes apple trees, grapes, plums, cabbage, eggplants, cucumbers, wheat, rice, soybeans, rye and many other crops.

One of the main areas in which genetic engineering technologies are used is agriculture. A classic method for improving the quality of agricultural products is selection - a process in which, through artificial selection, individual plants or animals with certain properties are isolated and crossed for the hereditary transmission of these properties and their enhancement. This process is quite lengthy and not always truly effective. Genetic engineering has the ability to endow a living organism with properties that are uncharacteristic for it, to enhance the manifestation of some existing properties or to exclude them. This occurs through the introduction of new or exclusion of old genes from the body's DNA.

For example, a special variety of potato resistant to the Colorado potato beetle was developed in this way. To do this, the gene of the soil Thuringian bacillus Bacillus thuringiensis was introduced into the potato genome, which produces a special protein that is harmful to the Colorado potato beetle, but harmless to humans. The use of genetic engineering to change the properties of plants, as a rule, is done precisely to increase their resistance to pests, unfavorable environmental conditions, and improve their taste and growth qualities. Intervention in the genome of animals is used to accelerate their growth and increase productivity. In this way, the amount of essential amino acids and vitamins in agricultural products is also artificially increased, as well as their nutritional value.

The number of arguments for the use of GMF significantly outweighs the possible arguments against. Thus, supporters of GMF refer in particular to the high level of quality control of all genetically modified products (GMP). Over the twenty-year history of the use of these products in different countries of the world, not a single fact of their negative impact on human health has been revealed, which cannot be said about the products of traditional agriculture, in which the use of various types of fertilizers is inevitable, many of which are recognized as harmful to humans. Moreover, selection, which has been used in agriculture for centuries, essentially aims at the same genetic modification of organisms, only it does this over a much longer period of time. Genetic engineering is simply capable of introducing the necessary changes into the body in a short period of time, and therefore the use of GMF is no more dangerous than the use of any other products bred by classical selection.

Opponents of the use of genetic engineering in agriculture appeal to the lack of research on the safety of GMOs (however, this issue continues to be constantly studied), as well as the fact that GMOs sometimes cause the extinction of certain species. For example, feral genetically modified organisms can displace populations of wild species due to greater adaptability to unfavorable conditions environment.

3.2. Pharmaceuticals and medicine.

The production and use of vaccines against viral diseases allowed doctors to completely eliminate epidemics of plague and smallpox, from which millions of people previously died. The genetic engineering method, unlike other methods, makes it possible to obtain an absolutely harmless (not containing an infectious agent) vaccine. Work is also underway to produce vaccines against influenza, hepatitis and other human viral diseases.

The services of genetic engineering are especially successfully used by pharmacists, for whom this method produces relatively cheap but vital hormones, such as insulin, interferon, growth hormones and others of a protein nature. At the request of pharmacists, genetic engineers have established the production of the human hormone insulin (instead of the previously used animal insulin), which plays an important role in the fight against diabetes. The method of genetic engineering also produces fairly cheap and pure human interferon, a protein with a universal antiviral effect, an antigen of the hepatitis B virus.

Currently, Escherichia coli (E. coli) has become a supplier of such important hormones as insulin and somatotropin. Previously, insulin was obtained from animal pancreatic cells, so its cost was very high. To obtain 100 g of crystalline insulin, 800-1000 kg of pancreas is required, and one gland of a cow weighs 200 - 250 grams. This made insulin expensive and difficult to access for a wide range of diabetics. In 1978, researchers from Genentech first produced insulin in a specially engineered strain of Escherichia coli. Insulin consists of two polypeptide chains A and B, 20 and 30 amino acids long. When they are connected by disulfide bonds, native double-chain insulin is formed. It has been shown that it does not contain E. coli proteins, endotoxins and other impurities, does not produce side effects like animal insulin, and is no different from it in biological activity. Subsequently, proinsulin was synthesized in E. coli cells, for which a DNA copy was synthesized on an RNA template using reverse transcriptase. After purifying the resulting proinsulin, it was split into native insulin, while the stages of extraction and isolation of the hormone were minimized. From 1000 liters of culture fluid, up to 200 grams of the hormone can be obtained, which is equivalent to the amount of insulin secreted from 1600 kg of the pancreas of a pig or cow.

Somatotropin is a human growth hormone secreted by the pituitary gland. A deficiency of this hormone leads to pituitary dwarfism. If somatotropin is administered in doses of 10 mg per kg of body weight three times a week, then in a year a child suffering from its deficiency can grow 6 cm. Previously, it was obtained from cadaveric material, from one corpse: 4 - 6 mg of somatotropin in terms of final pharmaceutical product. Thus, the available quantities of the hormone were limited, in addition, the hormone obtained by this method was heterogeneous and could contain slow-growing viruses. In 1980, the Genentec company developed a technology for the production of somatotropin using bacteria, which was devoid of these disadvantages. In 1982, human growth hormone was obtained in culture of E. coli and animal cells at the Pasteur Institute in France, and in 1984, industrial production of insulin began in the USSR. In the production of interferon, both E. coli, S. cerevisae (yeast), and a culture of fibroblasts or transformed leukocytes are used. Safe and cheap vaccines are also obtained using similar methods.

Practical application. Now they are able to synthesize genes, and with the help of such synthesized genes introduced into bacteria, a number of substances are obtained, in particular hormones and interferon. Their production constituted an important branch of biotechnology. Interferon, a protein synthesized by the body in response to a viral infection, is now being studied as a possible treatment for cancer and AIDS. It would take thousands of liters of human blood to produce the amount of interferon that just one liter of bacterial culture provides. It is clear that the benefits from mass production of this substance are very large. Insulin, obtained on the basis of microbiological synthesis, which is necessary for the treatment of diabetes, also plays a very important role. Genetic engineering has also been used to create a number of vaccines that are now being tested to test their effectiveness against the human immunodeficiency virus (HIV), which causes AIDS. With the help of recombinant DNA, human growth hormone is also obtained in sufficient quantities, the only means of treating a rare childhood disease - pituitary dwarfism. Another promising direction in medicine associated with recombinant DNA is the so-called. gene therapy. In these works, which have not yet left the experimental stage, a genetically engineered copy of a gene encoding a powerful antitumor enzyme is introduced into the body to fight a tumor. Gene therapy has also begun to be used to combat inherited disorders in the immune system. In agriculture, dozens of food and feed crops have been genetically modified. In livestock farming, the use of biotechnologically produced growth hormone has increased milk yield; A vaccine against herpes in pigs was created using a genetically modified virus.

4. Cloning.

The basis for the emergence of one of the most promising biomedical areas in cell replacement therapy - therapeutic cloning - were two most important discoveries the end of the 20th century. This is, firstly, the creation of a cloned sheep Dolly, and secondly, the production of embryonic stem cells (ESCs ).

Cloning is the reproduction of a living being by its non-reproductive (somatic) cells. Cloning of organs and other organs is the most important task in the field of transplantology, traumatology and other areas of medicine and biology. When transplanting cloned organs, there are no rejection reactions and there are no possible adverse consequences (for example, cancer developing against the background of immunodeficiency). Cloned organs are a salvation for people who have been in car accidents or other disasters, as well as those in need of radical help due to any diseases. Cloning could make it possible for childless people to have their own children, help people suffering from severe genetic diseases. So, if the genes that determine any hereditary disease are contained in chromosomes, then the nucleus of her own somatic cell is transplanted into the mother’s egg, then a child will appear, devoid of dangerous genes, a copy of the mother. If these genes are contained in the mother's chromosomes, the nucleus of the father's somatic cell will be transferred to her egg and a healthy child, copy of father. The further progress of mankind is largely connected with the development of biotechnology. At the same time, it must be taken into account that the uncontrolled spread of genetically engineered living organisms and products can disrupt the biological balance in nature and pose a threat to human health.

Cloning an entire organism is called reproductive cloning. Research in this direction is still underway, but there has been some progress.

The case of cloning the sheep Dolly in Great Britain is widely known. This mammal cloning experiment was carried out by a group of scientists led by Jan Wilmut. Then nuclei taken from the udder of a donor animal were transferred into 277 eggs. Of these, 29 embryos were formed, one of which survived. Dolly was born on July 5, 1996 and became the first mammal to be successfully cloned. The cloned animal lived for 6.5 years and died on February 14, 2003 from a progressive lung disease caused by a retrovirus. It is reported to be a common disease in sheep kept indoors, and Dolly was rarely taken out to graze for safety reasons.

There are some misconceptions about cloning. Thus, cloning a person or animal is definitely not capable of replicating consciousness. The cloned individual will not be endowed with the intelligence of the original organism; he will need upbringing, education, etc. Moreover, the question of the complete external identity of the clone is also controversial. As a rule, a clone is not a complete copy of the original, because When cloning, only the genotype is copied, which does not mean an unambiguous repetition of the organism’s phenotype. The phenotype is formed on the basis of certain genetic data, however, the conditions in which the clone will be grown can in some way affect its development: height, weight, physique, and some features of mental development.

In most countries of the world, any work on human reproductive cloning is prohibited. Such human cloning faces even greater ethical, religious and legal problems than therapeutic cloning. In principle, there is no definite public opinion on this matter, just as the world’s largest religions are not able to give an unambiguous assessment of this phenomenon, because it goes beyond the scope of their classical teachings and therefore requires argumentation. Some legal difficulties also arise, such as issues of paternity, maternity, inheritance, marriage and some others. The development of cloning is also unsafe for reasons of control over it, as well as the possible leakage of technology to criminal and terrorist circles. Of particular concern is the high percentage of failures in cloning, which poses the danger of the emergence of deformed people.

4.1 State of research on therapeutic cloning in Russia.

Despite the boom about the great potential of ESCs in the treatment of various diseases, work on therapeutic cloning is practically not being carried out in Russia. This is primarily due to the lack of a legislative framework for conducting research using human oocytes and embryos. With the adoption of such laws, there is a real opportunity for Russia to develop therapeutic cloning very quickly. Our country has effective cellular technologies for obtaining reconstructed embryos using nuclear transplantation. Essentially, the foundations of modern technologies for nuclear transfer of somatic cells, combining microsurgery and electrofusion, were first developed here in the 80s of the last century. There are also effective technologies for obtaining human ESC lines.

It is possible to implement the tasks of therapeutic cloning on the basis of reproduction centers, which, in addition to their direct purpose, can become centers for obtaining ESC lines, first of all, directly for female patients of this center and any members of their families. It can be expected that with the development of therapeutic technologies, obtaining one’s own ESCs will become available to every person. It is necessary to carry out close cooperation between reproduction centers and relevant research laboratories focused on solving fundamental problems and developing new technologies. Similar technologies include reconstruction of embryos using non-invasive optical-laser micromanipulation techniques for the purpose of therapeutic cloning

5. Problems of genetic engineering.

Genetic engineering is a completely new technology that breaks down fundamental genetic barriers not only between species, but also between people, animals and plants. By combining genes from dissimilar and unrelated species, forever changing their genetic codes, new organisms are created that will pass on genetic changes to their descendants. Today, scientists are able to cut, paste, recombine, transform, edit and program genetic material. Animal and even human genes are added to plants or animals, giving rise to unimaginable transgenic life forms. For the first time in history, human beings became the architects of life. Bioengineers will be able to create tens of thousands of new organisms over the next few years. The prospects are frightening. Genetic engineering raises unprecedented ethical and social issues, and also threatens the well-being of the environment, human and animal health and the future of agriculture. The following describes just some of the problems associated with genetic engineering:

Genetically modified organisms that escape or are released from a laboratory can cause environmental destruction. Genetically engineered "biological pollutants" have the potential to be more destructive than even chemical pollutants. Because they are living, genetically modified foods are inherently more unpredictable than chemical ones—they can reproduce, migrate, and mutate. Once these genetically modified organisms are released into the environment, it will be almost impossible to return them back to the laboratory. Many scientists warn that releasing such organisms into external environment can lead to irreversible destructive consequences for the environment.

Genetic changes are likely to lead to unexpected results and dangerous surprises. Biotechnology is an imprecise science, and scientists can never guarantee 100 percent success. Serious cases have occurred in practice. Researchers conducting experiments at Michigan State University recently found that genetically altering plants that are resistant to viruses can cause viruses to mutate into new, more dangerous forms or forms that can attack other plant species. Other scary scenarios: Foreign genes from genetically modified plants can be transferred along with pollen, insects, wind or rain to other crops, as well as wild and weed plants. Disaster can happen if properties of genetically modified crops, such as resistance to viruses or insects, are acquired by weeds, for example. Genetically modified plants can produce toxins and other substances that can harm birds and other animals. Genetic engineering of plants and animals will almost certainly endanger species and reduce biological diversity. Due to their "superior" genes, some of the GIs of plants and animals will inevitably go out of control, conquering wild species. This has already happened when exotic species were imported into the country; for example, in North America there were problems with Dutch elm disease and climbing pueraria. What will happen to wild species, for example, when scientists release carp, salmon, or trout into the environment that are twice the size and eat twice as much food as their wild relatives? Another danger lies in the creation of new types of crops and domestic animals. Once scientists create what is called the “perfect tomato” or “perfect chicken,” they will be reproduced in large quantities; “less desirable” species will be left by the wayside. "Perfect" animals and plants would then be cloned (reproduced as exact genetic copies), further reducing the base of available genes on the planet.

Genetic changes in crops and animals can trigger the development of toxic and allergic reactions in people. A person with an allergy to nuts or shellfish, for example, would have no way of knowing whether a tomato or other food has been altered to include proteins from the allergenic food, and consuming these GI foods could have fatal consequences. Alternatively, genetic engineers could take protein from bacteria found in the soil, the ocean—anywhere—and add it to human food. Such substances have never been added to food before, so there is no information about their toxicity and allergenicity.

There are known cases where genetically modified foods have caused harm to people. In 1989 and 1990, genetically engineered L-tryptophan, a common dietary supplement, killed more than 30 Americans and permanently disabled more than 5,000 with the potentially fatal and painful blood disorder eosinophilia-myalgia syndrome before being banned. The manufacturer, Showa Denko K.K., Japan's third-largest chemical company, used a genetically modified bacterium to create this over-the-counter supplement. It is believed that the bacterium was somehow infected through the process of DNA recombination. Products do not indicate that they have been genetically modified. The patenting of GI products and the widespread production of biotechnology products will destroy farming as it has been known since ancient times. If this trend is not stopped, the patenting of transgenic plants and animals by the meat and dairy industry will soon lead to the development of lease farming, where farmers will lease plants and animals from biotech conglomerates and pay for the seeds and offspring. Ultimately, over the next few decades, agriculture will be wiped out and taken over by industrial biosynthesis factories controlled by chemical and biotech companies. Never again will people enjoy natural, fresh food. Hundreds of millions of farmers and other workers around the world will lose their earnings. The sustainable agricultural system will be destroyed.

Genetic modification and patenting of animals will reduce the status of living beings to industrial products and lead to greater suffering. In January 1994, it was announced that the complete genome map of cows and pigs had been elucidated, which preceded further development of animal experiments. In addition to the inherent cruelty of such experiments (faulty specimens were born with painful defects, lame, blind, etc.), these "production" creatures had no greater value for their “creators” than mechanical inventions. Animals genetically engineered for use in laboratories, such as the infamous “Harvard mouse,” which had a human cancer-causing gene that was passed on to all subsequent generations, were designed to suffer. A purely reductionist science, biotechnology reduces the significance of life to pieces of information (genetic code) that can be disassembled and reassembled as one pleases. Stripped of their uniqueness and intimacy, animals that are mere objects for their “inventors” will be treated as such. Patents for more than 200 genetically altered "bizarre" animals are currently pending approval.

Genetically engineered organisms have never been adequately or properly tested for safety. To date, there is no appropriate government organization created to deal with this radical new class of creatures, which potentially pose enormous threats to health and the environment. The US Food and Drug Administration's policy on genetically modified foods illustrates the problem. In May 1992, this country developed a new policy regarding biotechnological products: genetically modified products will not be considered separately from natural ones; they will not be tested for safety; they will not contain a label indicating that they have been genetically modified; The US government will not track GI foods. As a result, neither the government nor consumers will know which whole or processed foods have been genetically modified. Vegetarians and people who exclude certain foods from their diets due to religious beliefs face the prospect of unwittingly consuming vegetables and fruits containing genetic material from animals and even humans. And the health consequences will only be determined through trial and error - by consumers.

By patenting the genes and living organisms they discover, a small corporate elite will soon control the entire genetic heritage of the planet. Scientists who “discover” genes and ways to manipulate them can receive patents—and thus ownership—not just of the genetic modification technologies, but also of the genes themselves. Chemical, pharmaceutical and biotechnology companies such as DuPont, Upjohn, Bayer, Dow, Monsanto, Cib-Geigy and Rhone-Poulenc are urgently trying to identify and patent genes from plants and animals. and people to make a complete takeover of the agriculture, livestock and manufacturing industries food products. These are the same companies that once promised a carefree life with pesticides and plastic. Can their plans for the future be trusted?

Studying the human genome could lead to the declassification of personal information and new levels of discrimination. Some people are already being denied health insurance based on “bad” genes. Will employers require gene scanning, and will they refuse jobs to their employees based on the results? Will the government gain access to our personal genetic profiles? It's easy to imagine new level discrimination against those whose genetic profiles indicate that they are, for example, less intelligent or predisposed to certain diseases.

Genetic engineering has already been used to “improve” the human race, a practice called eugenics. Gene scanning already allows us to find out whether the fetus carries the genes for certain hereditary diseases. Will we soon begin to dispose of fetuses on the basis of non-life-threatening defects such as myopia, homosexuality, or purely cosmetic reasons? Researchers at the University of Pennsylvania have applied for a patent to GI animal sperm cells so that the properties passed on from one generation to the next can be altered; this suggests that the same is possible with humans. The transition from animal eugenics to human eugenics is just one small step. Everyone wants the best for their children, but where do we stop? Inadvertently, we may soon repeat the Nazis' efforts to create a "perfect" race.

The US military is creating an arsenal of genetically modified biological weapons. Although the creation of offensive biological weapons has been made illegal under international treaties,The US continues to develop such weapons for defense purposes. However, genetically modified biological agents are identical whether they are used for defense or attack. Areas of research for such weapons include the following: bacteria resistant to all antibiotics; more resistant and dangerous bacteria and viruses that live longer and kill faster, as well as new organisms that can invalidate the effect of a vaccine or reduce the natural resistance of people and plants. The possibility of developing pathogens that can disrupt a person's hormonal balance enough to cause death, and transforming harmless bacteria (such as those found in the human gut) into killers, has also been explored. Some experts believe that GI pathogens are also being developed that target specific racial groups.

Not all scientists are optimistic about genetic engineering. Among the skeptics is Irwin Chargoff, a distinguished biochemist often called the father of molecular biology. He warns that not all innovation results in “progress.” Chargoff once called genetic engineering a "molecular Auschwitz" and warned that genetic engineering technology puts the world at greater risk than the advent of nuclear technology. “I feel that science has crossed a barrier that should remain unbroken,” he wrote in his autobiography. Noting the "terrifying irreversibility" of the planned genetic engineering experiments, Chargoff warned that "... you cannot undo new uniform life... it will outlive you, and your children, and your children's children. An irreversible attack on the biosphere is something so unheard of, so unimaginable to previous generations, that I can only wish that I was not to blame for it.”

5. Conclusion

Public opinion. Despite the obvious benefits of genetic research and experimentation, the very concept of “genetic engineering” has given rise to various suspicions and fears, and has become a subject of concern and even political controversy. Many fear, for example, that some virus that causes cancer in humans will be introduced into a bacterium that normally lives in the body or on the skin of a person, and then this bacterium will cause cancer. It is also possible that a plasmid carrying a drug resistance gene will be introduced into a pneumococcus, causing the pneumococcus to become resistant to antibiotics and causing pneumonia to become untreatable. These kinds of dangers undoubtedly exist. Genetic research is conducted by serious and responsible scientists, and methods to minimize the possibility of accidental spread of potentially dangerous microbes are constantly being improved. In assessing the possible dangers that these studies pose, they should be compared with the true tragedies caused by malnutrition and diseases that kill and maim people.

Genetic engineering is one of the most actively developing and promising technologies of our time, which in the future will be able to solve many issues in medicine and more. My personal opinion on most controversial genetic engineering issues leans towards allowing research and application of these technologies.

In my opinion, genetic modification of organisms, with reasonable control over this process, can solve some serious problems modernity. In particular, the use of genetic modification in medicine for the treatment of various diseases seems to me to be a positive phenomenon that does not cause any complaints at this stage of the development of science.

As for the use of genetic modification in agriculture and the distribution of genetically modified products, in my opinion, their hypothetical danger to human health is not actually confirmed. It seems to me that if the standard safety studies of these products indicate that their use is possible, then they do not need any additional research. GMOs in this case should be considered as a new type of plant or product and, provided that it meets all standard food safety standards, its use should be clearly permitted. I also share the point of view that GMF, due to special control over them, improvement of their properties at the genetic level and the absence of the need to use various fertilizers harmful to humans during cultivation, can be even safer than regular products agriculture.

Cloning issues present serious ethical issues when it comes to human cloning. At this stage, the arguments about the need for reproductive cloning of people, in my opinion, are not convincing enough, and therefore the ban on reproductive cloning seems justified to me. However, this does not mean that all research in this area should be stopped, because if science can provide a greater likelihood of clone survival, and the public can resolve other controversial issues, reproductive cloning may well be allowed.

The issue of therapeutic cloning is also quite complicated, because in order to obtain stem cells it is necessary to stop the development of an embryo, which, in principle, can develop into a child. It seems to me that this ethical problem is in some way similar to the problem of abortion. However, all things considered, I am inclined to advocate allowing therapeutic cloning because this can save a person’s life at the cost of a possible life interrupted at the birth stage.

As for the study and research of cloning issues itself, in particular the issues of reproductive cloning of animals, in my opinion, it should be allowed, since it is unreasonable to prohibit it in the context of the use of animals in any other types of laboratory research.

List of used literature.

1. Bochkarev A. I. Concepts modern natural science: textbook for university students / A. I. Bochkarev, T. S. Bochkareva, S. V. Saksonov; edited by prof. A. I. Bochkareva. – Togliatti: TGUS, 2008. – 386 p.

2. G96Guseikhanov M.K., Radzhabov O.R. Concepts of modern natural science: Textbook. - 6th ed., revised. and additional - M.: Publishing and trading corporation "Dashkov and Co", 2007. - 540 p.

- This is the production of products and materials necessary for humans using living organisms, cultured cells and biological processes.

Biotechnology opportunities are unusually large due to the fact that its methods are more profitable than conventional ones: they are used for optimal conditions(temperature and pressure), more productive, environmentally friendly and do not require chemical reagents that poison the environment, etc.

Biotechnology objects: numerous representatives of groups of living organisms - microorganisms (viruses, bacteria, protists, yeast, etc.), plants, animals, as well as cells and subcellular structures isolated from them (organelles). Biotechnology based on the physiological and biochemical processes occurring in living systems, as a result of which energy is released, the synthesis and breakdown of metabolic products, and the formation of chemical and structural components of the cell.

Main directions of biotechnology:

1) production with the help of microorganisms and cultured eukaryotic cells of biologically active compounds (enzymes, vitamins, hormonal drugs), medications (antibiotics, vaccines, serums, highly specific antibodies, etc.), as well as proteins, amino acids used as feed additives;

2) application biological methods anti-pollution ( biological treatment wastewater, soil pollution, etc.) and to protect plants from pests and diseases;

3) creation of new useful strains of microorganisms, plant varieties, animal breeds, etc.

Objectives, methods and achievements of biotechnology.

Humanity needs to learn how to effectively change the hereditary nature of living organisms in order to provide itself with good quality food and raw materials and at the same time not lead the planet to environmental disaster. Therefore it is no coincidence main task breeders in our time have become the solution to the problem of creating new forms of plants, animals and microorganisms that are well adapted to industrial methods of production, can withstand unfavorable conditions, effectively use solar energy and, most importantly, make it possible to obtain biologically pure products without excessive environmental pollution. Fundamentally new approaches A solution to this fundamental problem is the use of genetic and cellular engineering in breeding.

Genetic (genetic) engineering -

a branch of molecular genetics associated with the targeted creation of new DNA molecules capable of multiplying in a host cell and controlling the synthesis of the necessary cell metabolites.

Having emerged at the intersection of nucleic acid chemistry and microbial genetics, genetic engineering deals with deciphering the structure of genes, their synthesis and cloning, inserting genes isolated from the cells of living organisms or newly synthesized genes into plant and animal cells in order to specifically change their hereditary properties.

To carry out gene transfer (or transgenesis) from one species of organism to another, often very distant in origin, it is necessary to perform several complex operations:

isolation of genes (individual DNA fragments) from cells bacteria, plants or animals. In some cases, this operation is replaced by artificial synthesis of the necessary genes;

connection (stitching) individual DNA fragments of any origin into a single molecule as part of a plasmid;

introduction of hybrid plasmid DNA containing the desired gene into the host cells;

copying (cloning) of this gene in the new host to ensure its operation.

Cloned genes are microinjected into mammalian eggs or plant protoplasts (isolated cells lacking a cell wall) and whole animals or plants are grown from them, into whose genome the cloned genes are built (integrated). Plants and animals whose genomes have been altered through genetic engineering operations are called transgenic plants or transgenic animals.

Transgenic mice, rabbits, pigs, sheep have already been obtained, in the genome of which foreign genes of various origins operate, including genes of bacteria, yeast, mammals, humans, as well as transgenic plants with genes of other, unrelated species. Transgenic organisms indicate the great potential of genetic engineering as an applied branch of molecular genetics (for example, a new generation of transgenic plants has been obtained, which are characterized by such valuable traits as resistance to herbicides, insects, etc.).

Today, genetic engineering methods have made it possible carry out synthesis in industrial quantities of hormones such as insulin, interferon and somatotropin (growth hormone), which are necessary for the treatment of a number of human genetic diseases - diabetes mellitus, certain types of malignant tumors, dwarfism,

Using genetic methods, strains of microorganisms (Ashbya gossypii, Pseudomonas denitrificans, etc.) were also obtained that produce tens of thousands of times more vitamins (C, B 3, B 13, etc.) than the original forms.

Cell engineering-

a set of methods used to construct new cells. Includes cultivation and cloning of cells in specially selected media, cell hybridization, transplantation of cell nuclei and other microsurgical operations for “disassembly” and “assembly” (reconstruction) of viable cells from individual fragments.

At the core cell engineering lies the use of methods cultivation isolated cells and tissues on an artificial nutrient medium under controlled conditions. This became possible due to the ability of plant cells, as a result of regeneration, to form a whole plant from a single cell. Regeneration conditions have been developed for many cultivated plants - potatoes, wheat, barley, corn, tomatoes, etc. Working with these objects makes it possible to use non-traditional methods of cell engineering in breeding - somatic hybridization, haploidy, cell selection, overcoming uncrossability in culture, etc.

Cloning -

a method of producing several identical organisms through asexual (including vegetative) reproduction. In this way, many species of plants and animals have reproduced in nature for millions of years. However, now the term "cloning" is usually used in a narrower sense and means copying cells, genes, antibodies and even multicellular organisms in the laboratory. Specimens that appear as a result of asexual reproduction are, by definition, genetically identical, however, hereditary variability can be observed in them, caused by random mutations or created artificially by laboratory methods.

Thematic assignments

A1. The production of drugs, hormones and other biological substances is carried out in such a direction as

1) genetic engineering

2) biotechnological production

3) agricultural industry

4) agronomy

A2. When is tissue culture most useful?

1) when obtaining a hybrid of apple and pear

2) when breeding pure lines of smooth-seeded peas

3) if necessary, transplant skin to a person with a burn

4) when obtaining polyploid forms of cabbage and radish

A3. In order to artificially produce human insulin using genetic engineering methods on an industrial scale, it is necessary

1) introduce the gene responsible for the synthesis of insulin into bacteria, which will begin to synthesize human insulin

2) introduce bacterial insulin into the human body

3) artificially synthesize insulin in a biochemical laboratory

4) grow a culture of human pancreatic cells responsible for the synthesis of insulin.

Biotechnology is the conscious production of products and materials needed by humans using living organisms and biological processes.

Since time immemorial, biotechnology has been used mainly in the food and light industries: in winemaking, bakery, fermentation of dairy products, in the processing of flax and leather, based on the use of microorganisms. In recent decades, the possibilities of biotechnology have expanded enormously. This is due to the fact that its methods are more profitable than conventional ones for the simple reason that in living organisms, biochemical reactions catalyzed by enzymes occur under optimal conditions (temperature and pressure), are more productive, environmentally friendly and do not require chemical reagents that poison the environment.

The objects of biotechnology are numerous representatives of groups of living organisms - microorganisms (viruses, bacteria, protozoa, yeasts), plants, animals, as well as cells isolated from them and subcellular components (organelles) and even enzymes. Biotechnology is based on physiological and biochemical processes occurring in living systems, which result in the release of energy, synthesis and breakdown of metabolic products, and the formation of chemical and structural components of the cell.

The main direction of biotechnology is the production, using microorganisms and cultured eukaryotic cells, of biologically active compounds (enzymes, vitamins, hormones), medications (antibiotics, vaccines, serums, highly specific antibodies, etc.), as well as valuable compounds (feed additives, for example, essential amino acids, feed proteins, etc.). Genetic engineering methods have made it possible to synthesize in industrial quantities hormones such as insulin and somatotropin (growth hormone), which are necessary for the treatment of human genetic diseases.

One of the most important areas of modern biotechnology is also the use of biological methods to combat environmental pollution (biological treatment of wastewater, contaminated soil, etc.).

Thus, to extract metals from wastewater, bacterial strains capable of accumulating uranium, copper, and cobalt can be widely used. Other bacteria of the genera Rhodococcus and Nocardia are successfully used for emulsification and sorption of petroleum hydrocarbons from the aquatic environment. They are capable of separating water and oil phases, concentrating oil, purifying waste water from oil impurities. By assimilating petroleum hydrocarbons, such microorganisms convert them into proteins, B vitamins and carotenes.

Some of the halobacteria strains are successfully used to remove fuel oil from sandy beaches. Genetically engineered strains have also been obtained that can break down octane, camphor, naphthalene, and xylene and effectively utilize crude oil.

The use of biotechnology methods to protect plants from pests and diseases is of great importance.

Biotechnology is making its way into heavy industry, where microorganisms are used to extract, convert and process natural resources. Already in ancient times, the first metallurgists obtained iron from bog ores produced by iron bacteria, which are capable of concentrating iron. Now methods have been developed for the bacterial concentration of a number of other mineral metals: manganese, zinc, copper, chromium, etc. These methods are used to develop dumps of old mines and poor deposits, where traditional mining methods are not economically viable.

Genetic engineering is one of the most important methods of biotechnology. It involves the targeted artificial creation of certain combinations of genetic material capable of functioning normally in a cell, i.e., multiplying and controlling the synthesis of final products. There are several types of genetic engineering method, depending on the level and characteristics of its use.

Genetic engineering is used mainly on prokaryotes and microorganisms, although it has recently begun to be used on higher eukaryotes (for example, plants). This method involves the isolation of individual genes from cells or the synthesis of genes outside cells (for example, based on messenger RNA synthesized by a given gene), directed rearrangement, copying and propagation of isolated or synthesized genes (gene cloning), as well as their transfer and inclusion in the subject to change genome. In this way, it is possible to achieve the inclusion of “foreign” genes in bacterial cells and the synthesis of compounds important for humans by bacteria. Thanks to this, it was possible to introduce the insulin synthesis gene from the human genome into the E. coli genome. Insulin synthesized by bacteria is used to treat patients with diabetes.

The development of genetic engineering became possible thanks to the discovery of two enzymes - restriction enzymes, which cut the DNA molecule in strictly defined areas, and ligases, which stitch the pieces together various molecules DNA with each other. In addition, genetic engineering is based on the discovery of vectors, which are short circular DNA molecules that independently reproduce in bacterial cells. With the help of restriction enzymes and ligases, the required gene is inserted into the vectors, subsequently achieving its inclusion in the genome of the host cell.

Cell engineering is a method of constructing a new type of cells based on their cultivation, hybridization and reconstruction. It is based on the use of cell and tissue culture methods. There are two areas of cell engineering: 1) the use of cells transferred into culture for the synthesis of various compounds useful for humans; 2) the use of cultured cells to obtain regenerated plants from them.

Plant cells in culture are an important source of valuable natural substances, since they retain the ability to synthesize their characteristic substances: alkaloids, essential oils, resins, biologically active compounds. Thus, ginseng cells transferred into culture continue to synthesize, as in the composition of the whole plant, valuable medicinal raw materials. Moreover, in culture, any manipulations can be carried out with cells and their genomes. Using induced mutagenesis, it is possible to increase the productivity of cultured cell strains and carry out their hybridization (including distant hybridization) much easier and simpler than at the level of the whole organism. In addition, genetic engineering work can be carried out with them, as with prokaryotic cells.

By hybridizing lymphocytes (cells that synthesize antibodies, but grow reluctantly and for a short time in culture) with tumor cells that have potential immortality and are capable of unlimited growth in an artificial environment, one of the most important problems of biotechnology has been solved. modern stage- hybridoma cells capable of endless synthesis of highly specific antibodies of a certain type were obtained.

Thus, cell engineering makes it possible to construct a new type of cells using the mutation process, hybridization and, moreover, to combine individual fragments of different cells (nuclei, mitochondria, plastids, cytoplasm, chromosomes, etc.), cells of various types, related not only to different genera, families, but also kingdoms. This facilitates the solution of many theoretical problems and has practical significance.

Cell engineering is widely used in plant breeding. Hybrids of tomato and potato, apple and cherry have been developed. Plants regenerated from such cells with altered heredity make it possible to synthesize new forms and varieties that have beneficial properties and resistant to adverse environmental conditions and diseases. This method is also widely used to “rescue” valuable varieties affected by viral diseases. From their sprouts in culture, several apical cells are isolated, not yet affected by the virus, and healthy plants are regenerated from them, first in a test tube, and then transplanted into soil and propagated.



Cell engineering

Cell engineering– growing cells outside the body on special nutrient media, where they grow and multiply, forming a tissue culture. This is a method for constructing a new type of cells based on their cultivation, hybridization and reconstruction. Cellular reconstruction associated with the creation of a viable cell from individual fragments of different cells (nucleus, cytoplasm, chromosomes, etc.). With the help of cell engineering, it is possible to connect the genomes of very distant species. The fundamental possibility of fusion of somatic animal cells with plant cells is shown. The study of hybrid cells makes it possible to solve many theoretical problems of biology and medicine: to clarify the mutual influences of the nucleus and cytoplasm; mechanisms of cell differentiation and regulation of cell reproduction, transformation of normal cells into cancer cells, etc.

At hybridization artificially combine whole cells (cell protoplasts) to form hybrid genome. Using enzymes or ultrasound, the cell walls of plant cells are removed and the “naked” cell protoplasts are connected. After this, the cell walls are restored and form callus- an unorganized cell mass, causing differentiation of the cells of which a whole hybrid plant is obtained.

Cell engineering is widely used in biotechnology, for example, the use hybrids(hybrid cells) to produce monoclonal antibodies. Based on genetically modified cells, it is possible to create new forms of plants that have useful traits and are resistant to favorable environmental conditions and diseases.

Genetic engineering – artificial genome rearrangement. A branch of molecular genetics associated with the targeted creation in vito (in vitro) of new combinations of genetic material capable of multiplying in a host cell and synthesizing metabolic products. Accompanied by the artificial transfer of necessary genes from one type of living organism (bacteria, plants, animals) to another, often distant in origin. Modern gene technologies are used for gene therapy, i.e. treatment of hereditary diseases by introducing “healthy” genes to a person.

The highest achievement of modern biotechnology is genetic transformation, the transfer of foreign genes and other material carriers of heredity into the cells of plants, animals and microorganisms, the production of transgenic organisms with new or enhanced properties and characteristics. In terms of its goals and capabilities in the future, this direction is strategic. It makes it possible to solve fundamental problems in the selection of biological objects for stability, high productivity and product quality while improving the environmental situation in all types of production. However, to achieve these goals, enormous difficulties must be overcome in increasing the efficiency of genetic transformation and, above all, in identifying genes, creating their cloning banks, deciphering the mechanisms of polygenic determination of traits and properties of biological objects, ensuring high gene expression and creating reliable vector systems. Already today, in many laboratories around the world, including in Russia, using genetic engineering methods, fundamentally new transgenic plants, animals and microorganisms have been created that have received commercial recognition.

Modern biotechnology

Modern biotechnology closely interfaces with a number of scientific disciplines, carrying out their practical application or being their main tool (Fig. 1).

Rice. 1. Relationship of biotechnology with other sciences (according to V.I. Kefeli, 1989)

In molecular biology, the use of biotechnological methods makes it possible to determine the structure of the genome, understand the mechanism of gene expression, model cell membranes in order to study their functions, etc. The construction of the necessary genes using genetic and cellular engineering methods makes it possible to control the heredity and vital activity of animals, plants and microorganisms and create organisms with new properties useful to humans that have not previously been observed in nature.

The microbiology industry currently uses thousands of strains of different microorganisms. In most cases, they are improved by induced mutagenesis and subsequent selection. This allows large-scale synthesis of various substances.

Some proteins and secondary metabolites can only be produced by culturing eukaryotic cells. Plant cells can serve as a source of a number of compounds - atropine, nicotine, alkaloids, saponins, etc. Animal and human cells also produce a number of biologically active compounds. For example, pituitary cells contain lipotropin, a stimulator of fat breakdown, and somatotropin, a hormone that regulates growth.

Continuous animal cell cultures have been created that produce monoclonal antibodies, widely used for diagnosing diseases. In biochemistry, microbiology, and cytology, methods for the immobilization of both enzymes and whole cells of microorganisms, plants and animals are of undoubted interest. In veterinary medicine, biotechnological methods such as cell and embryo culture, in vitro oogenesis, and artificial insemination are widely used. All this indicates that biotechnology will become a source not only of new food products and medicines, but also of energy and new chemical substances, as well as organisms with desired properties.