GENETIC ENGINEERING(syn. genetic engineering) - 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. At the heart of G. and. the possibility of purposeful manipulation with fragments of nucleic acids due to the latest achievements of molecular biology and genetics lies. These achievements include the establishment of the universality of the genetic code (see), that is, the fact that in all living organisms the inclusion of the same amino acids in a protein molecule is 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 separate genes or fragments of nucleic acids in an isolated form, to carry out in vitro synthesis of fragments of nucleic acids to - t, to combine the obtained fragments into a single whole. Thus, change of hereditary properties of an organism by means of G. and. is reduced to the construction of new genetic material from various fragments, the introduction of this material into the recipient organism, the creation of conditions for its functioning and stable inheritance.
One of the ways to obtain genes is chem. synthesis. After Holly (A. Holli) in the USA, A. A. Baev in the USSR and other researchers managed to decipher the structure of various transport RBGK (tRNA), X. Koran et al., carried out a chem. synthesis of DNA encoding baker's yeast alanine tRNA.
But the most effective method of artificial gene synthesis is associated with the use of the enzyme RNA-dependent DNA polymerase (reverse transcriptase) discovered by Baltimore (D. Baltimore) and Temin (H. Temin) in oncogenic viruses (see). This enzyme has been isolated and purified from cells infected with certain RNA-containing oncogenic viruses, including avian myeloblastosis virus, Rous sarcoma, and mouse leukemia. Reverse transcriptase provides DNA synthesis on the messenger RNA (mRNA) template. The use of mRNA molecules as templates for DNA synthesis greatly facilitates the artificial synthesis of individual structural genes of higher organisms, since the sequence of nitrogenous bases in an mRNA molecule is an exact copy of the sequence of nitrogenous bases of the corresponding structural genes, and the technique for isolating various mRNA molecules is quite well developed. Advances in isolating globin protein mRNA, which is part of human, animal and bird hemoglobin, eye lens protein mRNA, immunoglobin mRNA, mRNA of a specific malignant tumor (myeloma) protein, made it possible to synthesize the structural part of the genes encoding some of these proteins using reverse transcriptase.
However, in the body, structural genes function together with regulatory genes, the nucleotide sequence of which is not reproduced by the mRNA molecule. Therefore, none of these methods allows the synthesis of a set of structural and regulatory genes. The solution to this problem became possible after the development of methods for isolating individual genes. To isolate bacterial genes, small DNA-containing cytoplasmic structures are used that can replicate (see Replication) independently of the bacterial chromosome. These structures form a single group of extrachromosomal genetic elements of bacteria - plasmids (see Plasmids). Some of them can be introduced into the bacterial chromosome, and then spontaneously or under the influence of inducing agents, for example. UV irradiation, move from the chromosome to the cytoplasm, taking with it the adjacent chromosomal genes-cells of the host. Extrachromosomal genetic elements of bacteria with such properties are called episomes [F. Jacob, Wollman (E. Wollman)]. Episomes (see) include moderate phages (see. Bacteriophage), sex factor of bacteria, drug resistance factors of microorganisms (see), bacteriocinogenic factors (see). In the cytoplasm, genes captured by episomes replicate in their composition and often form many copies. The development of an effective method for isolating plasmids, in particular temperate phages carrying the genetic material of the bacterial chromosome, and isolating a fragment of the bacterial cell chromosome included in the bacteriophage genome made it possible in 1969 for J. Beckwith et al. to isolate the lactose operon, a group of genes that control synthesis enzymes necessary for the absorption of lactose by Escherichia coli. A similar technique was used to isolate and purify the gene that controls the synthesis of Escherichia coli tyrosine transfer RNA (see Ribonucleic acids).
The use of plasmids makes it possible to obtain practically any bacterial genes in isolated form, and, consequently, the possibility of constructing DNA molecules from various sources. Such hybrid structures can be accumulated in cells in significant quantities, since many plasmids under certain conditions intensively replicate in the bacterial cytoplasm, forming tens, hundreds, and even thousands of copies.
G.'s successes and. associated with the development of techniques for combining genetic structures from different sources in a single DNA molecule. The decisive factor in the design of hybrid molecules in vitro was the use of restriction endonucleases - special enzymes capable of cutting DNA molecules in strictly defined areas. Such enzymes are found in Escherichia coli cells carrying R-type plasmids, which make bacteria resistant to certain drugs, in cells of Haemophilus influenzae, Serratia marcescens and other microorganisms. One of the most commonly used enzymes of this type is the EcoRI restriction endonuclease, which is synthesized by the RI plasmid in E. coli cells. The enzyme recognizes a section of DNA with a unique sequence of six base pairs and cuts the double-stranded DNA structure in this section so that single-stranded ends of four nucleotides are formed on both sides (so-called sticky ends). Since the enzyme cuts DNA molecules, regardless of their origin, in a strictly defined way, all DNA fragments resulting from the action of the enzyme will have the same sticky ends. Complementary sticky ends of any DNA fragments are combined by hydrogen bonds, forming a hybrid circular DNA (Fig.). To stabilize the hybrid DNA molecule, another enzyme is used - polynucleotide ligase, which restores covalent bonds broken by the restriction enzyme. The sequence specifically recognized by EcoRI occurs in DNA no more than 4,000-16,000 base pairs apart. Therefore, a DNA fragment formed under the action of EcoRI may include at least one gene undamaged by the enzyme (on average, one gene contains 1000–1500 base pairs).
The use of restriction endonucleases and a number of other enzymes makes it possible to obtain complex recombinant DNA. A group of researchers in the United States led by P. Berg managed to combine genetic information from three sources as part of a single DNA molecule: the complete genome (see) of the oncogenic monkey virus SV40, part of the genome of the temperate bacteriophage λ and the group of E. coli genes responsible for the assimilation galactose. The designed recombinant molecule was not tested for functional activity, because the authors of this work stopped before the potential danger of the spread of oncogenic animal viruses in the bacterial population living in the human intestine. It is known that the purified DNA of viruses can penetrate into various mammalian cells and be stably inherited by them.
For the first time, functionally active hybrid DNA molecules were constructed in the USA by S. Cohen et al. Cohen's group consistently solved the problem of combining and cloning (selective accumulation) of DNA molecules isolated from species increasingly phylogenetically distant from each other. The cloning procedure usually consists in the fact that DNA from various sources is fragmented using restriction endonucleases, then these fragments are combined in vitro into a common structure and introduced into the recipient organism, which in Cohen's experiments is Escherichia coli. It has been established that cells of several bacterial species (including Escherichia coli, Salmonella typhimurium, Staphylococcus aureus) can be transformed (see Transformation) using recombinant DNA molecules. In this case, the plasmid part of the hybrid molecule (or one of the plasmids, if two plasmids from different sources are combined in the hybrid molecule) serves as a vector, i.e., ensures the transfer of phylogenetically alien genetic material to recipient cells and its reproduction in them. The first plasmid used by Cohen et al. as a vector was the plasmid pSC101 obtained by him in vitro, which controls the resistance of bacteria to tetracycline. This small plasmid is only 8000 bp long. It is attacked by the EcoRI enzyme in only one site, and the enzyme does not damage the ability of the plasmid to subsequently replicate in E. coli cells and control tetracycline resistance. These features made it possible to use it for the construction of hybrid DNA molecules in vitro. At the first stages, plasmid DNA isolated from various bacterial species and then from higher organisms was attached to pSC101. Thus, “chimeric” plasmids (that is, not capable of occurring in natural conditions) were created, which combined in their composition the genetic material of Escherichia coli, a DNA segment from oocytes of the clawed frog Xenopus laevis, which controls the synthesis of ribosomal RNA, and a DNA segment of a sea urchin that controls synthesis of histone proteins, or mouse mitochondrial DNA. In the cells of Escherichia coli, into which such hybrid, "chimeric" plasmids were introduced, the work of the genes of higher organisms was registered.
Unlike pSC101, which is present in the cell only in 4-6 copies, some other plasmids used as vectors can replicate many times under certain conditions, forming several thousand copies in one cell. Such properties are possessed, for example, by the ColEI plasmid, which controls the synthesis of colicin (see Bacteriocinogeny). Like pSC101, ColEI is cleaved by the EcoRl enzyme in only one site, and foreign DNA, also treated with EcoRI, is easily attached to the resulting linear molecule with sticky ends. Thus, the genes of the tryptophan operon of Escherichia coli were "sewn" to ColEI. In cells carrying many copies of the constructed hybrid plasmid, the production of enzyme proteins controlled by tryptophan biosynthesis genes increased dramatically. In the in vitro system, it was possible to attach the ColEI plasmid to certain R-factors and a temperate phage. Such work was first performed in the USSR under the guidance of Academician A. A. Baev and Professor S. I. Alikhanyan. Combined vector plasmids formed by ColEI and R-factors are able to multiply intensively in bacterial cells, like ColEI, and at the same time determine the resistance of cells to antibiotics, which greatly simplifies the selection of bacteria - carriers of hybrid plasmids.
Temperate phages are also used as vectors. In the in vitro system, hybrid bacteriophage particles were constructed that included bacterial genes, DNA of other phages or higher organisms (for example, DNA of the fruit fly Drosophila) in their structure.
The functional activity of hybrid DNA is determined by the possibility of their transfer into the cells of recipient organisms and subsequent multiplication (amplification) in these cells. As recipients, not only bacteria, as mentioned above, but also cells of higher organisms are already being effectively used, so far, however, only in the form of a tissue culture cultivated outside the body. There are indications that the DNA of phages carrying bacterial genes can penetrate into human connective tissue cells (fibroblasts), into protoplasts, or into an undifferentiated culture (callus) of plant cells. In 1971, Amer. researcher Merrill (S. R. Merril) et al., reported on experiments to correct a hereditary defect - galactosemia (see) by introducing into "sick" cells of galactose genes of bacteria included in the DNA of the transducing phage. As a result, the cells of a patient with galactosemia, defective in the enzyme beta-D-galactose-1-phosphate uridyltransferase, unable to assimilate galactose, restored their normal ability to grow in the presence of galactose, and previously absent enzymatic activity was registered in their extracts. A similar result was obtained by Horst (J. Horst) et al, with the introduction of a bacterial gene that controls the synthesis of beta-galactosidase in the fibroblasts of a patient with generalized gangliosidosis, characterized by a severe deficiency of this enzyme. Manion (W. Munyon) and his collaborators. using the herpes virus, they transferred the gene that controls the synthesis of thymidine kinase from human cells to mouse cells, restoring the ability of defective mouse fibroblasts to synthesize this enzyme.
One of the ways to transfer genetic information in the culture of human, animal and plant cells is the hybridization of somatic cells, developed by Ephrussi (V. Ephrussi) and Barsky (G. Barski). The effectiveness of this method has improved significantly since it was found that particles of inactivated Sendai-type parainfluenza virus increase the frequency of cell fusion from a wide variety of sources. The possibility of transferring individual genes from isolated Chinese hamster chromosomes into mouse connective tissue cells has been demonstrated. Hybrids of human and mouse cells are described, in which part of the human chromosomes is removed, while the other part remains functionally active. The development of cell microsurgery methods made it possible to transplant cell nuclei from somatic cells into fertilized eggs and, as a result, obtain absolutely identical organisms. Cell hybridization made it possible to induce the synthesis of human globin in frog germ cells. All these examples demonstrate G.'s potential and.
Practical value of G. and. for medicine is associated with the prospects for correcting hereditary metabolic defects in humans (see Gene therapy), creating microorganisms that have lost their pathogenicity, but retained the ability to form immunity, the synthesis of antibiotics, amino acids, hormones, vitamins, enzymes, immunoglobulins, etc., based on the use of microorganisms that have included the corresponding genes. Exceptional results can be obtained in the near future G. and. plants. With the help of G.'s methods and. they are trying to create plants that can absorb atmospheric nitrogen and improve the protein composition of plant foods. The successful solution of these problems will dramatically increase the productivity of plants, reduce the production and consumption of mineral nitrogen, and thereby significantly improve the environment (see). The possibility of creating completely new forms of animals and plants by overcoming interspecific barriers of interbreeding is being studied. However at G.'s assessment and. as a new form of mastering wildlife, one should take into account not only its possible revolutionary role in biology, medicine and agriculture, but also the opportunities arising in connection with its development for the emergence of new forms of pathogenic microorganisms, the danger of the spread of hybrid DNA in populations of bacteria living in humans, carrying Oncogenic viruses, etc. Of course, the deliberate use of the achievements of science, including G. and., for inhumane, misanthropic purposes is possible only in a society in which the good of man is sacrificed to profit and aggression.
From additional materials
Genetic engineering continues to be a rapidly advancing research method in molecular biology and genetics. It should be noted that the concepts of "genetic engineering" and "genetic engineering" are not completely synonymous, since research related to genetic engineering is not limited to manipulations with genes as such. Currently, genetic engineering methods allow for the most in-depth and detailed analysis of natural nucleic acids - substances responsible for the storage, transmission and implementation of genetic information (see Nucleic acids.), As well as create modified or completely new ones that are not found in nature. genes (see gene), combinations of genes and express them with high efficiency in a living cell (see gene expressivity). Of the specific practical achievements of genetic engineering in the last decade, the most important should be the creation of producers of biologically active proteins - insulin (see), interferon (see), growth hormone (see Somatotropic hormone), etc., as well as the development of genetic engineering methods activation of those links of a metabolism, to-rye are connected with formation of low-molecular biologically active agents. In this way, producers of certain antibiotics, amino acids and vitamins are obtained, many times more effective than the producers of these substances, derived by traditional methods of genetics and selection. Methods are being developed for obtaining pure protein vaccines against hepatitis, influenza, herpes, and foot-and-mouth disease viruses, the idea of using vaccination with vaccinia virus has been implemented, in the genome of which genes encoding the synthesis of proteins of other viruses (for example, hepatitis or influenza viruses) are embedded: as a result Inoculations with a virus constructed in this way, the body develops immunity not only against smallpox, but also against hepatitis, influenza or other diseases caused by that virus, the protein to-rogo is encoded by the built-in gene.
The world collection of restriction endonucleases - restrictases, the main "tools" of genetic engineering manipulations, has grown significantly. More than 400 restrictases "recognizing" apprx. 100 specific sites (sites) of different structure in DNA molecules (see Deoxyribonucleic acids) and splitting the DNA polynucleotide chain at these sites. With the help of one such enzyme or a combination of several restriction enzymes, almost any gene can be isolated as part of one or more DNA fragments (so-called restriction fragments). This has expanded the possibilities of genetic engineering not only in relation to the isolation of genes, but also in relation to the activation of their work, the analysis of the structure of genes and their molecular environment. Methods for the synthesis of whole genes with a given sequence of nucleotides have been developed, it has become possible to supply synthesized and natural genes with various regulatory nucleotide sequences, replace, insert, delete single nucleotides in strictly specified sections of a gene, shorten or complete its nucleotide chain with an accuracy of one nucleotide.
The achievement of genetic engineering was its penetration into the organization and functioning of the mechanisms of heredity in the cells of higher organisms, including humans. It is on higher eukaryotes that the most interesting data have been obtained using genetic engineering methods. The success of genetic engineering is largely associated with the production of new specialized vectors that allow efficient cloning (propagation) of individual DNA fragments (genes) and synthesizing proteins encoded by these genes.
Restriction fragments connected to DNA vectors are cloned in a living cell using the ability of such vectors to reproduce (replicate) in a cell in multiple copies. Depending on the size of the fragments to be cloned and the purpose of the study, vectors of one of four types are used - plasmids (see), phages (see. Bacteriophage), cosmids or derivatives of phages with single-stranded DNA.
For cloning relatively small DNA fragments (up to 10 thousand base pairs), plasmid vectors (pBR322, pAT 153, pUR250, pUC19, etc.) are used. The achievement of genetic engineering in recent years was the production of vectors based on phage X (Charon 4A, gtwes-B), in which part of the genome was replaced by a fragment of foreign DNA. The hybrid genome is artificially "packed" into a protein coat and bacteria are infected with this reconstructed phage. Forming several thousand copies in the cell during reproduction, the reconstructed phage lyses it and is released into the culture medium. With the help of such vectors, DNA fragments of 10-25 thousand base pairs are cloned.
Cosmid vectors (pIB8, MUA-3) are a hybrid of phage X and a plasmid. They contain the so-called COS sequences of phage DNA required for packaging phage genomes into a protein shell, and a segment of plasmid DNA that allows cosmid vectors to replicate in bacteria in the same way as plasmids do. Thus, the resulting recombinant genome infects bacteria with high efficiency like a bacteriophage, but multiplies in them like a plasmid without causing the death of a bacterial cell. Cosmids are used for cloning DNA fragments up to 35-45 thousand base pairs long.
The vectors, which are derivatives of phages with single-stranded DNA (M13 mp8, M13, mp73, etc.), are constructed on the basis of the circular DNA molecule of the M13 bacteriophage. For embedding foreign DNA, a replicative double-stranded phage DNA molecule is used. A vector carrying a foreign DIC is introduced into bacterial cells, where the recombinant molecules multiply without lysing this cell and "bud off" into the culture medium as a viral particle with a single-stranded DNA molecule. These vectors are used to clone DNA fragments (up to 300-400 base pairs).
The gene required for genetic engineering manipulations is obtained by cloning the appropriate recombinant DNA molecules and selecting such clones. In those cases when the genes of higher organisms and humans are cloned / expression to-rykh in E. coli (most often used for such purposes) is impossible, the cloning and selection procedure is carried out in several stages. At the first stage, a so-called a library of genes from DNA fragments (cloned directly from the cell genome) or from cloned DNA copies (cDNA) of the corresponding messenger RNA. Comparing the structure of fragments of genomic DNA and the corresponding cDNA, they obtain important information about the organization of the genetic material, and in the case of hereditary diseases, about the nature of the anomalies in the genetic material, the consequence of which is this disease. From the gene library, using modern techniques, it is possible to extract the required gene with the surrounding genome regions. At present, complete libraries of genes of many microorganisms, plants and animals (up to mammals and humans) have been created. Several hundred genes and other nucleotide sequences in human DNA have already been cloned and to some extent studied.
The possibilities of genetic engineering research are not limited to cloning a gene and obtaining a large number of its copies. It is often necessary not only to clone a gene, but also to ensure its expression in a cell, i.e., to implement the information contained in it into the amino acid sequence of the polypeptide chain of the protein encoded by this gene. If a gene introduced into a bacterial cell is obtained from bacteria of the same (or close) species, then it may be sufficient to isolate the gene with regulatory elements that control its expression. However, with a few exceptions, the regulatory nucleotide sequences of evolutionarily distant organisms are not interchangeable. Therefore, in order to achieve, for example, the expression of a eukaryotic gene in E. coli cells, the regulatory region is removed from it, and the structural part of such a gene is attached (at a certain distance) to the regulatory region of the bacterial gene. Significant progress in the development of this technique was achieved after the discovery of the Ba131 nuclease enzyme, which has the unique property of hydrolyzing both chains of a double-stranded linear DNA molecule starting from the end of the molecule, i.e. this enzyme removes “extra” nucleotide sequences of any length from the end of the DNA fragment . Currently, the structural and regulatory regions are isolated separately using those restrictases, the “recognition” sites of which are located most successfully on the polynucleotide chain, then the “extra” nucleotide sequences are removed and the structural region of the eukaryotic gene is connected to the regulatory region of the bacterial gene. In this way, it is possible to achieve not only the expression of eukaryotic genes in bacterial cells, but, conversely, bacterial genes in the cells of higher and lower eukaryotes.
The success of genetic engineering is closely related to the development and improvement of methods for determining the nucleotide sequence (sequencing) in DNA molecules. A significant number of restrictases at the disposal of researchers makes it possible to isolate certain DNA fragments with absolute specificity, and the development and improvement of cloning methods makes it possible to obtain fragments of even unique genes in quantities necessary for analysis. DNA sequencing methods have proven to be so effective that often, by determining the DNA nucleotide sequence, data are obtained on the nucleotide sequence in the corresponding RNA molecules and on the sequence of amino acid residues in the synthesized protein molecule. When processing the results of DNA sequencing, computers are widely used. For a more complete and faster interpretation of the obtained experimental data, national and international computer "banks" of nucleotide sequences are being created. At present, the complete nucleotide sequences of the genomes of a number of bacterial plasmids and viruses have been determined, and the problem of determining the complete nucleotide sequences of first individual chromosomes, and then the entire genome of higher organisms, including humans, is already being solved.
With the help of genetic engineering methods, deviations in the structure of certain sections of human genes were found, which was the cause of hereditary diseases. Most often, this method is the so-called. b lot analysis. The isolated cellular DNA is subjected to restriction enzyme hydrolysis, the resulting fragments are separated by size using agarose or polyacrylamide gel electrophoresis. The separated fragments are transferred ("reprinted") onto specially treated chromatographic paper, nitrocellulose or nylon filter and again subjected to electrophoretic separation. Cut out places of electropherograms corresponding to individual fractions and containing the same type of DNA fragments; cut sections of electrophoregrams are incubated with a previously cloned gene or part of it, or with a chemically obtained one. synthesis by a nucleotide sequence containing a radioactive label. The marked DNA contacts only those fragments of the analyzed cellular DNA, to-rye have sequences of nucleotides complementary to it. A change in the distribution and amount of a fixed label compared to the norm makes it possible to judge rearrangements in the analyzed gene or nucleotide sequences adjacent to it.
The sites of "recognition" of certain restrictases in the DNA molecule are unevenly distributed, therefore, during hydrolysis by these enzymes, the DNA molecule is split into a number of fragments of various lengths. The rearrangement of the DNA structure, as a result of which the existing “recognition” sites disappear or appear, leads to a change in the set of these fragments (the so-called restriction fragments), i.e., to the appearance of restriction fragment length polymorphism (GVDRF). Rearrangements in the DNA molecule may or may not cause changes during synthesis or in the structure of the encoded protein; rearrangements that do not cause changes are the majority, and they cause a normal RFLP. It turned out that RFLP is a clear genetic trait. Currently, RFLP analysis has become one of the most accurate methods used in human genetics and medical genetics. For a number of hereditary diseases, forms of RFLP are described that directly indicate the presence of a disease or the carriage of a pathologically altered gene.
Genetic engineering marked the beginning of a new direction of research, called "genetics in reverse." The traditional genetic analysis (see) is carried out in the following sequence: the sign is chosen, the link of a sign with a genetic determinant and localization of this determinant in relation to already known is established. In reverse genetics, everything happens in the reverse order: a DNA fragment with an unknown function is selected, the linkage of this DNA fragment with other regions of the genome and its connection with certain traits is established. This approach made it possible to develop methods for early diagnosis and detection of carriers of such diseases as Huntington's chorea, Duchenne's disease, cystic fibrosis, the biochemical nature of hereditary defects in which is not yet known. Using the genealogical method for establishing the patterns of hereditary transmission of Huntington's chorea, it was shown that the G8 DNA fragment isolated from the human genome is closely linked to the gene that determines the disease, and the shape of the RFLP G8 fragment in this population can diagnose this disease and identify carriers of defective genes.
There are still many technical difficulties on the way of introducing the methods used in genetic engineering into medical practice. Many laboratories around the world are actively developing practically suitable genetic engineering diagnostic methods, and it is hoped that such methods will find application in the near future, if not for mass genetic screening (screening) during the medical examination of the population, then, at least, for a sample survey of high-risk groups for hereditary diseases.
Genetic engineering makes it possible not only to copy natural compounds and processes, but also to modify them and make them more efficient. An example of this is a new line of research called protein engineering. Calculations made on the basis of data on the amino acid sequence and the spatial organization of protein molecules show that with certain replacements of certain amino acid residues in the molecules of a number of enzymes, a significant increase in their enzymatic activity is possible. In an isolated gene encoding the synthesis of a particular enzyme, strictly controlled replacement of certain nucleotides is carried out by genetic engineering methods. During the synthesis of an enzymatic protein under the control of such a modified gene, a pre-planned replacement of strictly defined amino acid residues in the polypeptide chain occurs, which causes an increase in enzymatic activity many times over compared to the activity of a natural prototype.
In the field of agriculture, genetic engineering is expected to make a major contribution to the selection of new high-yielding plant varieties that are resistant to drought, diseases, and pests, as well as to the development of new highly productive crop varieties. animals.
Like any achievement of science, the successes of genetic engineering can be used not only for the benefit, but also to the detriment of humanity. Specially conducted studies have shown that the danger of uncontrolled spread of recombinant DNA is not as great as previously thought. Recombinant DNA and bacteria carrying them turned out to be very unstable to environmental influences, not viable in humans and animals. It is known that in nature and without human intervention there are conditions that provide an active exchange of genetic information, this is the so-called. gene flow. However, nature has created many effective barriers to the penetration of alien genetic information into the body. At present, it is obvious that when working with most recombinant DNA molecules, the usual precautions are quite sufficient, to-rye are used, for example, by microbiologists when working with infectious material. For special cases, effective methods have been developed for both biological protection and physical isolation of experimental objects from humans and the environment. Therefore, the very strict first versions of the rules for working with recombinant DNA were revised and significantly softened. As for the deliberate use of the achievements of genetic engineering to the detriment of humans, both scientists and the public must actively fight to ensure that this danger remains only theoretically possible.
See also Biotechnology.
Bibliography: Alikhanyan S. I. Successes and prospects of genetic engineering, Genetics, vol. 12, Jvft 7, p. 150, 1976, bibliogr.; AlikhanyanS. I. et al. Obtaining functioning recombinants (hybrid) DNA molecules, in vitro, ibid., vol. I, No. 11, p. 34, 1975, bibliogr.; Baev A. A. Genetic engineering, Priroda, M1, p. 8, 1976; Tikhomirova L.P. and others. Hybrid DNA molecules of phage X and plasmids ColEl, Dokl. USSR Academy of Sciences, vol. 223, no. 4, p. 995, 1975, bibliogr.; Brown D.D.a. S t e r n R. Methods of gene isolation, Ann. Rev. Biochem., v. 43, p. 667, 1974, bibliogr.; C h a n g A. C. Y. a. o. Studies of mouse mitochondrial DNA in Escherichia coli, Cell, v. 6, p. 231.1975, bibliogr.; Hedgpeth J., Goodman H. M. a. B o y e r H. W. DNA nucleotide sequence restricted by the R1 endonuclease, Proc. nat. Acad. sci. (Wash.), v. 69, p. 3448, 1972, bibliogr.; Hershfield V. a. o. Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA, ibid., v. 71, p. 3455, 1974; Morrow J. F. a. o. Replication and transcription of eukaryotic DNA in Escherichia coli, ibid., p. 1743; T e m i n H. M. a. Mizu-tani S. RNA-dependent DNA polymerase in virions of Rous sarcoma virus, Nature (Lond.), v. 226, p. 1211, 1970.
Biotechnology, ed. A. A. Baeva, M., 1984; B about h to about in N. P., Zakharov A. F. and Ivanov V. I. Medical genetics, M., 1984; M a n i a-tis G., FrichE. and Sambrook J. Methods of genetic engineering. Molecular cloning, trans. from English, M., 1984; A n t o n a r a k i s S. E. a. o. DNA polymorphism and molecular pathology of human globin gene clusters, Hum. Genet., v. 69, p. 1, 1985; Beaudet A. L. Bibliography of cloned human and other selected DNAs, Amer. J. hum. Genet., v. 37, p. 386, 1985; In o t s t e i n D. a. o. Construction of a genetic linkage map in man using restriction fragment length polymorphisms, ibid., v. 32, p. 314, 1980; G u s e 1 1 a J. E. a. o. DNA markers for nervous system diseases, Science, v. 225, p. 1320, 1984; Motulsky A. G. Impact of genetic manipulation on society and medicine, ibid., v. 219, p. 135, 1983; White R. a. o. A closely linked genetic marker for cystic fibrosis, Nature (Lond.), v. 318, p. 382, 1985; Wo o S. L. C., L i d s to y A. S. a. Guttler F. Prenatal diagnosis of classical phenylketonuria by gene mapping, J. Amer. med. Ass., v. 251, p. 1998, 1984.
L. S. Chernin, V. H. Kalinin.
genetic engineering
Modern biology fundamentally differs from traditional biology not only in the greater depth of development of cognitive ideas, but also in a closer connection with the life of society, with practice. We can say that in our time, biology has become a means of transforming the living world in order to meet the material needs of society. This conclusion is illustrated, first of all, by the close relationship between biology and biotechnology, which has become the most important area of material production, an equal partner of man-made mechanical and chemical technologies, as well as medicine.
Since its inception, biology and biotechnology have always developed together, and from the very beginning, biology has been the scientific basis of biotechnology. However, for a long time, the lack of own data did not allow biology to have a very large impact on biotechnology. The situation changed dramatically with the creation in the second half of the 20th century. genetic engineering methodology, which is understood as genetic manipulation with the aim of constructing new and reconstructing existing genotypes. Being a methodical achievement by its nature, genetic engineering did not lead to a breakdown of the prevailing ideas about biological phenomena, did not affect the basic provisions of biology, just as radio astronomy did not shake the basic provisions of astrophysics, the establishment of the "mechanical equivalent of heat" did not lead to a change in the laws of heat conduction, and the proof The atomistic theory of matter did not change the relations of thermodynamics, hydrodynamics and elasticity theory (A.A. Baev).
Nevertheless, genetic engineering has opened a new era in biology for the reason that new opportunities have appeared for penetrating into the depths of biological phenomena in order to further characterize the forms of existence of living matter, more effectively study the structure and function of genes at the molecular level, and understand the subtle mechanisms of work. genetic apparatus. Advances in genetic engineering mean a revolution in modern
natural science. They determine the criteria for the value of modern ideas about the structural and functional features of the molecular and cellular levels of living matter. Modern data on living things are of gigantic cognitive significance, because they provide an understanding of one of the most important aspects of the organic world and thus make an invaluable contribution to the creation of a scientific picture of the world. Thus, having sharply expanded its cognitive base, biology through genetic engineering also had a leading influence on the rise of biotechnology.
Genetic engineering creates groundwork on the path to understanding the ways and means of "designing" new or improving existing organisms, giving them great economic value and the ability to dramatically increase the productivity of biotechnological processes. However, genetic engineering has created new horizons for medicine in the field of diagnosis and treatment of many diseases, both non-hereditary and hereditary. She opened new avenues in the search for new drugs and materials used in medicine. Genetic engineering and biotechnology have stimulated the development of bionanotechnology methods.
Within the framework of genetic engineering, there are genetic and cellular engineering. Genetic engineering is the manipulation to create recombinant DNA molecules. This methodology is often referred to as molecular cloning, gene cloning, recombinant DNA technology, or simply genetic manipulation. It is important to emphasize that the object of genetic engineering are DNA molecules, individual genes. On the contrary, cell engineering is understood as genetic manipulations with isolated individual cells or groups of plant and animal cells.
GENETIC ENGINEERING AND ITS TOOLS
Genetic engineering is a set of various experimental techniques (methods) that provide construction (reconstruction), cloning of DNA molecules and genes with specified goals.
Genetic engineering methods are used in a certain sequence (Fig. 127), and several stages are distinguished in the execution
a typical genetic engineering experiment aimed at cloning a gene, namely:
1. Isolation of plasmid DNA from the cells of the organism of interest (initial) and isolation of the DNA vector.
2. Cutting (restriction) of the DNA of the original organism into fragments containing the genes of interest using one of the restriction enzymes and isolation of these genes from the restriction mixture. At the same time, the vector DNA is cut (restricted), turning it from a circular structure into a linear one.
3. Linking the DNA segment (gene) of interest to the vector DNA in order to obtain hybrid DNA molecules.
4. Introduction of recombinant DNA molecules by transformation into some other organism, for example, into E. coli or somatic cells.
5. Inoculation of bacteria, into which hybrid DNA molecules were introduced, on nutrient media allowing the growth of only cells containing hybrid DNA molecules.
6. Identification of colonies consisting of bacteria containing hybrid DNA molecules.
7. Isolation of cloned DNA (cloned genes) and its characterization, including sequencing of nitrogenous bases in the cloned DNA fragment.
Rice. 127.Successive stages of genetic engineering experiment
In the course of evolution, bacteria developed the ability to synthesize the so-called restriction enzymes (endonucleases), which became part of the cellular (bacterial) restriction-modification system. In bacteria, restriction-modification systems are the intracellular immune defense system against foreign DNA. Unlike higher organisms, in which the recognition and destruction of viruses, bacteria, and other pathogens occurs extracellularly, in bacteria, protection from foreign DNA (DNA of plants and animals in which they live) occurs intracellularly, i.e. when foreign DNA enters the cytoplasm of bacteria. In order to protect themselves, bacteria have also evolved the ability to “tag” their own DNA with methylating bases at specific sequences. For the same reason, foreign DNA, due to the absence of methyl groups in it on the same sequences, is melted (cut) into fragments by various bacterial restrictases, and then degraded by bacterial exonucleases to nuleotides. We can say that in this way bacteria protect themselves from the DNA of plants and animals, in whose organism they live temporarily (as pathogens) or permanently (as saprophytes).
Restriction enzymes were first isolated from E. coli in 1968. It turned out that they are able to cut (melt) DNA molecules at different sites (places) of restriction. These enzymes were called class I endonucleases. Then, class II endonucleases were found in bacteria, which specifically recognize restriction sites in foreign DNA and also carry out restriction at these sites. It is the enzymes of this class that began to be used in genetic engineering. At the same time, class III enzymes were discovered that melt DNA near recognition sites, but these enzymes are of no importance in genetic engineering.
The action of the restriction-modification system is "rationalized" by the so-called palindromic (recognizing) sequences of nitrogenous bases, which are DNA restriction sites. Palindromic sequences are sequences of bases that read the same forward and backward, such as the sequence of letters radar. Since the DNA strands have an antiparallel direction, a sequence is considered to be palindromic if it is identical when read in the direction from the 5" to the 3" end on the upper and on the lower strand from the 3" to the 5" end, namely :
Palindromes can be of any size, but most of the palindromes that are used as restriction enzyme recognition sites are 4, 5, 6, and rarely 8 bases long.
Restriction enzymes are an absolutely essential tool in genetic engineering for cutting out fragments (genes) of interest from large DNA molecules. Since more than 100 restriction enzymes are known, this allows the selection of restriction enzymes and the selective excision of fragments from the original DNA.
A remarkable feature of restrictases is that they produce cuts of molecules into several fragments (restrictions) of DNA in ledges, as a result of which one strand is longer than the other at the resulting ends, forming a kind of tail. Such ends (tails) are called "sticky" ends, as they are capable of self-complementarity.
Consider the results of restriction on the example of one of the most famous restrictases EcoRI from the restriction-modification system E. coi. Instead of melting the DNA at the center of the palindromic recognition sequence, this enzyme melts the DNA outside the center and produces 4 self-complementary (“sticky”) ends, consisting of a different number of nucleotides, namely:
These "sticky" ends are useful in genetic engineering because they can be reconnected complementarily at low temperatures, allowing efficient closure of DNA fragments.
Recognition sites and melting sites in the case of other restrictases have a different content, namely:
Following DNA restriction, restriction DNA fragments (DNA-restrictions) are isolated from the restriction mixture, which are then required for association with the vector. Restricted DNA is isolated using electrophoresis, since it is very easy to fractionate restricted DNA using this method due to the size of the restricted fragments and constant electrical charge-mass ratios. Fragments in an electric field migrate during electrophoresis at a frequency dependent on their size (mass). The larger (longer) the fragment, the slower it migrates in the electric field. The material in which electrophoresis is carried out is non-chargeable agarose or polyacrylamide. Ethidium bromide is used to identify the fragments, which stains the fragments, which leads to their easier detection.
The efficiency of electrophoresis is very high, since it can be used to separate fragments ranging in size from 2 to 50,000 bases.
After electrophoresis, fragments from agarose are isolated using various methods. Based on size comparison results
Restrictions of the same DNA, obtained using different restriction enzymes, build restriction maps, which show the restriction sites of each of the restriction enzymes used. In practical terms, restriction maps make it possible to determine not only the size of the restrictions, but also to find out the location of the loci of certain genes in DNA molecules.
Since in higher organisms, during transcription, heterogeneous DNA is synthesized, corrected by processing, in genetic engineering, complementary DNA (cDNA) is usually used, which is obtained by using mRNA as a template, on which reverse transcriptase synthesizes single-stranded DNA (cDNA), which is a copy of mRNA. Subsequently, these single-stranded DNAs are converted into double-stranded DNAs. Consider that cDNA contains continuous nucleotide sequences (transcribed and translated). It is cDNA that is used for restriction.
DNA fragments (restrictions) isolated after electrophoresis from agarose gels can be preliminarily subjected to sequencing; determine their nucleotide sequence. For this, chemical and enzymatic sequencing methods are used. The chemical method is based on obtaining fragments labeled with radioactive phosphorus (32 P) and removing one of the bases from these fragments, followed by taking into account the results of radioautography of gels containing these fragments. The enzymatic method is based on the fact that a nucleotide is introduced at the end of the analyzed fragment, which is then used in the synthesis of different fragments. in vitro, analyzed for the nucleotide sequence electrophoretically. To study specific nucleotide sequences in a DNA molecule, use
also hybridization of DNA-DNA, RNA-RNA, DNA-RNA, Northern-
and Southern blots.
Genetic vectors. The DNA segment (gene) that is intended for molecular cloning must be able to replicate when it is transferred into a bacterial cell, i.e. be a replica. However, he does not have this ability. Therefore, in order to ensure the transfer and detection of cloned genes in cells, they are combined with so-called genetic vectors. The latter must have at least two properties. First, vectors must be able to replicate
in cells, and at several ends. Secondly, they should allow the selection of cells containing the vector, i.e. possess a marker for which it is possible to counter-select cells containing the vector together with the cloned gene (recombinant DNA molecules). Plasmids and phages meet these requirements. Plasmids are good vectors because they are replicons and can contain genes for resistance to any antibiotic, which allows selection of bacteria for resistance to this antibiotic and, therefore, easy detection of recombinant DNA molecules.
(Fig. 128).
Rice. 128. Vector pBRl
Since there are no natural plasmid vectors, all plasmid vectors known so far have been artificially constructed. R-plasmids served as the starting material for the creation of a number of genetic vectors, in which excessive DNA sequences, including those with multiple restriction sites, were removed with the help of restrictases. This removal was determined by the fact that the plasmid vector should have only one recognition site for one restriction enzyme, and this site should lie in a functionally unimportant region of the plasmid genome. For example, the pBR 322 plasmid vector, which has ampicillin and tetracycline resistance genes, making it very convenient
for the selection of bacteria containing the cloned DNA segment, it has single restriction sites for more than 20 restriction enzymes, including such well-known restriction enzymes as Eco RI, Hind III, Pst I, Pva II and Sal I.
Phage vectors also have a number of advantages. They may include larger (longer) cloned DNA fragments compared to plasma vectors. Further, the transfer of the cloned fragment by phages into cells as a result of infection of the latter is more efficient than DNA transformation. Finally, phage vectors allow more efficient screening (recognition) on the agar surface of colonies containing cells carrying the cloned gene. Many phage vectors are based on the lambda phage.
In addition to phage, other viral vectors constructed on the basis of the herpes virus, as well as vectors constructed on the basis of yeast DNA, are also used.
If gene cloning is carried out using mammalian or plant cells, then the requirements for vectors are the same as in the case of cloning in bacterial cells.
Construction of recombinant DNA molecules. The direct construction of recombinant DNA molecules follows after the restriction of the studied DNA and vector DNA has been obtained. It consists in the closure of the restriction segments of the studied DNA with the vector DNA restriction, which, as a result of restriction, is transformed from circular to linear DNA.
To close the fragments of the DNA under study with the DNA of the vector, DNA ligase is used (Fig. 129). The ligation will be successful if the structures to be joined have 3'-hydroxyl and 5'-phosphate groups and if these groups are located in an appropriate relation to one another. Fragments are combined through their "sticky" ends as a result of self-complementarity. At high concentrations of fragments, the latter from time to time become in the correct position (opposite each other). Many restrictases, such as Eco RI, produce four-base "sticky" ends. The process of ligation of "sticky" ends, consisting of four bases, occurs at a low temperature (up to 12? C).
Rice. 129. DNA ligation
If fragments without "sticky" ends are formed during restriction, then they are "forcibly" converted into molecules with "sticky" ends using the transferase enzyme. This enzyme adds nucleotides to the 3" end of DNA. A poly-A tail can be added on one fragment, a poly-T tail on the other. Polymerase chain reaction (PCR) is also used to generate any desired DNA ends. The principle of PCR is based on the denaturation of DNA isolated from cells and its "annealing" with the addition of DNA oligonucleotides consisting of 15-20 nucleotides each to the renaturating chains. These oligonucleotides must be complementary to sequences in the chains separated by distances of 50-2000 nucleotides. DNA synthesis in vitro, they allow DNA polymerase to copy those regions that are between the "seeds". This copying gives a large number of copies of the studied DNA fragment.
Introduction of recombinant DNA molecules into cells. After the DNA fragment (gene) of interest is fused with a genetic vector using DNA ligase, the resulting recombinant molecules are introduced into cells in order to achieve their replication (due to the genetic vector) and increase the number of copies. The most popular way to introduce recombinant DNA molecules into cells, in which the vector is a plasmid, is transformation E. coli. For this purpose, bacterial cells are pre-treated with calcium or rubidium (ions), in order to
so that they become "competent" in the perception of recombinant DNA. To increase the frequency of DNA penetration into cells, the electroporation method is used, which consists in briefly exposing cells to an intense electric field. This treatment creates cavities in the cell membranes, which makes it easier for the cells to take up DNA. After the introduction of recombinant DNA molecules into bacteria, the latter are sown on MPA (meat-peptone agar) enriched with antibiotics to select the desired cells, i.e. cells containing recombinant DNA molecules. The frequency of transformation is low. Typically, one transformant occurs per 10 5 seeded cells. If the vector is phage, then transfection of cells (bacteria or yeast) with phage is resorted to. As for the somatic cells of animals, they are transfected with DNA in the presence of chemicals that facilitate the passage of DNA through plasma membranes. Direct microinjection of DNA into oocytes, cultured somatic cells, and mammalian embryos is also possible.
The most important point associated with molecular cloning is the search for a method to establish whether the cloned fragment is really included in the vector and, together with the vector, forming a recombinant DNA molecule, enters the cells. If we are talking about bacterial cells, then one of the methods is based on the consideration of insertional inactivation of the plasmid (vector) resistance gene. For example, in the plasmid vector pBR 322, which determines resistance to ampicillin and tetracycline, the only site for Pst I restriction enzyme is located in the locus occupied by the ampicillin resistance gene. Pst I melting at this site generates sticky ends allowing ligation of the cloned fragment to the vector DNA. However, in this case, the plasmid (vector) ampicillin resistance gene is inactivated, while the tetracycline resistance gene on the vector remains intact. It is the tetracycline resistance gene that is used to select cells transformed by recombinant DNA molecules. This makes it possible to verify that the cells of the grown colonies on the medium with tetracycline do indeed contain recombinant DNA molecules, they are checked using the so-called "spot test" on a pair of dishes with a solid medium, one of which contains ampicillin, while the other is devoid of this antibiotic. The DNA to be cloned is
only in tetracycline-resistant transformants. As for the transformants resistant to both ampicillin and tetracycline (ArTc), they contain plasmid (vector) molecules that spontaneously acquired a circular form without the inclusion of foreign (cloned) DNA in them.
Another method for detecting the insertion of foreign (cloned) fragments into a plasmid vector is based on the use of a vector containing the β-galactosidase gene. Insertion of foreign DNA into this gene inevitably inactivates the synthesis of β-galactosidase, which can be detected by seeding the transformed cells on a medium containing β-galactosidase substrates. This medium allows the selection of stained cell colonies. There are other methods as well.
As already noted, linear restriction fragments of vector DNA are capable of restoring the circular structure without including cloned segments in them. To reduce the frequency of spontaneous formation of such circular vector DNA molecules, the vector DNA restriction is treated with phosphatase. As a result, the formation of circular DNA molecules becomes impossible, since the ends of the 5'-PO 4 necessary for the action of the ligase will be absent.
The set of transformant colonies grown on a selective medium is a set of cells containing clones of different fragments (genes) of the cloned genomic or cDNA. Collections of these clones form the so-called DNA libraries, which are widely used in genetic engineering.
The final stage of gene cloning is the isolation and study of cloned DNA, including sequencing. Promising strains of bacteria or somatic cells containing recombinant DNA molecules that control the synthesis of proteins of interest that have commercial value are transferred to the industry.
CELL ENGINEERING
As noted at the beginning of the chapter, cell engineering refers to the genetic manipulation of isolated animal and plant cells. These manipulations are often in vitro, and their main goal is to obtain genotypes of these organisms with desired properties, primarily economically useful. As regards-
Xia man, then cell engineering was applicable to his germ cells.
A prerequisite for the development of cell engineering in humans and animals was the development of methods for cultivating their somatic cells on artificial nutrient media, as well as obtaining hybrids of somatic cells, including interspecific hybrids. In turn, advances in the cultivation of somatic cells have influenced the study of germ cells and fertilization in humans and animals. Since the 60s. 20th century Numerous experiments have been performed in several laboratories around the world on the transplantation of somatic cell nuclei into eggs artificially devoid of nuclei. The results of these experiments were often contradictory, but in general they led to the discovery of the ability of cell nuclei to ensure the normal development of the egg (see Chapter IV).
Based on the results of studying the development of fertilized eggs in the 60s. 20th century studies were also begun to ascertain the possibility of fertilization of eggs outside the mother's body. Very quickly, these studies led to the discovery of the possibility of fertilization of eggs with spermatozoa in vitro and the further development of the embryos formed in this way when implanted in the uterus of a woman. Further improvement of the methods developed in this area has led to the fact that the birth of "test-tube" children has become a reality. Already by 1981, 12 children were born in the world, whose life was given in the laboratory, in the test tube. At present, this section of cell engineering has become widespread, and the number of "test-tube" children is already tens of thousands (Fig. 130). In Russia, work on obtaining "test-tube" children was started only in 1986.
In 1993, a technique was developed for obtaining monozygotic human twins in vitro by dividing the embryos into blastomeres and growing the latter up to 32 cells, after which they could be implanted in the uterus of a woman.
Influenced by the results associated with test-tube babies, animals have also developed a technology called transplants embryos. It is associated with the development of a method for inducing poliovulation, methods for artificial fertilization of eggs and implantation of embryos in the body of animals - foster mothers. The essence of this technology is as follows:
schuschy. A highly productive cow is injected with hormones, resulting in poliovulation, which consists in the maturation of 10-20 cells at once. The eggs are then artificially fertilized with male reproductive cells in the oviduct. On the 7-8th day, the embryos are washed out of the uterus and transplanted into the uterus of other cows (foster mothers), which then give birth to twin calves. Calves inherit the genetic status of their original parents.
Rice. 130."Tube" children
Another area of cell engineering in animals is the creation of transgenic animals. The simplest way to obtain such animals is to introduce linear DNA molecules into the eggs of the original animals. Animals that develop from eggs so fertilized will carry a copy of the introduced gene in one of their chromosomes and, in addition, they will transmit this gene by inheritance. A more complex method for obtaining transgenic animals has been developed on mice that differ in coat color and is as follows. First, four-day-old embryos are removed from the body of a pregnant gray mouse and crushed into individual cells. Then the nuclei are extracted from the embryonic cells, they are transferred to the eggs of black mice, previously deprived of the nuclei. Black mouse eggs containing foreign nuclei are placed in test tubes
with nutrient solution for further development. Embryos developed from the eggs of black mice are implanted in the uterus of white mice. Thus, in these experiments, it was possible to obtain a clone of mice with a gray coat color, i.e. clone embryonic cells with desired properties. In Chapter IV, we examined the results of fertilization of artificially devoid of nuclei of sheep eggs with the nuclear material of somatic cells of animals of the same species. In particular, the nuclei were removed from the eggs of sheep, and then the nuclei of somatic cells (embryonic, fruit or cells of adult animals) were introduced into such eggs, after which the eggs fertilized in this way were introduced into the uterus of adult sheep. The born lambs turned out to be identical to the sheep donor. An example is Dolly the sheep. Clone calves, mice, rabbits, cats, mules and other animals have also been obtained. Such construction of transgenic animals is a direct way of cloning animals with economically useful traits, including individuals of a certain sex.
Transgenic animals were also obtained using source material belonging to different species. In particular, a method is known for transferring a gene that controls growth hormone from rats to mouse eggs, as well as a method for combining sheep blastomeres with goat blastomeres, which led to the emergence of hybrid animals (cows). These experiments indicate the possibility of overcoming species incompatibility at the earliest stages of development. Particularly tempting prospects open up (if species incompatibility is completely overcome) in the way of fertilization of the eggs of one species by the nuclei of somatic cells of another species. We are talking about the real prospect of creating economically valuable hybrids of animals that cannot be obtained by crossing.
It should be noted that nuclear transplantation work is not yet very effective. Experiments performed on amphibians and mammals have generally shown that their effectiveness is low, and it depends on the incompatibility between donor nuclei and recipient oocytes. In addition, the resulting chromosomal aberrations in transplanted nuclei in the course of further development, which are accompanied by the death of transgenic animals, are also an obstacle to success.
At the intersection of work on the study of cell hybridization and immunological studies, a problem arose associated with the production and study of so-called monoclonal antibodies. As noted above, the antibodies produced by the body in response to the introduction of an antigen (bacteria, viruses, red blood cells, etc.) are proteins called immunoglobulins and form a fundamental part of the body's defense system against pathogens. But any foreign body introduced into the body is a mixture of different antigens that will stimulate the production of different antibodies. For example, human erythrocytes have antigens not only for blood groups A (II) and B (III), but also many other antigens, including the Rh factor. Further, bacterial cell wall proteins or the capsid of viruses can also act as different antigens, causing the formation of different antibodies. At the same time, the lymphoid cells of the body's immune system are usually represented by clones. This means that even for this reason alone, in the blood serum of immunized animals, antibodies are always a mixture consisting of antibodies produced by cells of different clones. Meanwhile, for practical purposes, antibodies of only one type are needed; so-called monospecific sera, containing antibodies of only one type or, as they are called, monoclonal antibodies.
In search of methods for obtaining monoclonal antibodies, Swiss researchers in 1975 discovered a method of hybridization between mouse lymphocytes immunized with one or another antigen and cultured bone marrow tumor cells. Such hybrids are called "hybridoma". From the “lymphocytic” part, represented by a lymphocyte of one clone, a single hybridoma inherits the ability to cause the formation of the necessary antibodies, and of the same type, and thanks to the “tumor (myeloma)” part, it becomes capable, like all tumor cells, to multiply indefinitely on artificial nutrient media, giving a large population of hybrids. On fig. 131 shows a scheme for the isolation of cell lines synthesizing monoclonal antibodies. Monoclonal antibody-producing mouse cell lines are isolated by fusing myeloma cells with lymphocytes from the spleen of mice immunized five days previously.
desired antigen. Cell fusion is achieved by mixing them in the presence of polyethylene glycol, which induces the fusion of cell membranes, and then inoculating them on a nutrient medium that allows the growth and reproduction of only hybrid cells (hybridoma). Reproduction of hybridomas is carried out in a liquid medium, where they grow further and secrete antibodies into the culture liquid, and only one type, moreover, in unlimited quantities. These antibodies are called monoclonal. To increase the frequency of antibody formation, hybridoma cloning is resorted to, i.e. to the selection of individual colonies of hybridomas capable of producing the greatest amount of antibodies of the desired type. Monoclonal antibodies have found wide application in medicine for the diagnosis and treatment of a number of diseases. At the same time, the most important advantage of monoclonal technology is that it can be used to generate antibodies against materials that cannot be purified. On the contrary, it is possible to obtain monoclonal antibodies against cell (plasma) membranes of animal neurons. To do this, mice are immunized with isolated neuronal membranes, after which their splenic lymphocytes are combined with myeloma cells, and then proceed as described above.
Rice. 131. Obtaining monoclonal antibodies
GENETIC ENGINEERING AND MEDICINE
Genetic engineering turned out to be very promising for medicine, primarily in the creation of new technologies for obtaining physiologically active proteins used as drugs (insulin, somatostatin, interferons, somatotropin, etc.).
Insulin is used to treat people with diabetes, which is the third most common cause of death after heart disease and cancer. The world demand for insulin is several tens of kilograms. Traditionally, it is obtained from the pancreatic glands of pigs and cows, but the hormones of these animals are slightly different from human insulin. Pig insulin differs in one amino acid, while bovine insulin differs in three. It is believed that animal insulin often causes side effects. Although the chemical synthesis of insulin has been carried out for a long time, but until now the industrial production of hormones has remained very expensive. Now cheap insulin is obtained using a genetic engineering method by chemical-enzymatic synthesis of the insulin gene, followed by the introduction of this gene into E. coli, which then synthesizes the hormone. Such insulin is more "biological", as it is chemically identical to the insulin produced by cells of the human pancreas.
Interferons are proteins synthesized by cells mainly in response to infection of the body by viruses. Interferons are species specific. For example, in humans, there are three groups of interferons produced by various cells under the control of the corresponding genes. Interest in interferons is determined by the fact that they are widely used in clinical practice for the treatment of many human diseases, especially viral ones.
Having large sizes, interferon molecules are hardly available for synthesis. Therefore, most interferons are now obtained from human blood, but the yield with this method of obtaining is small. Meanwhile, the need for interferon is extremely high. This posed the challenge of finding an efficient method for the production of interferon in industrial quantities. Genetic engineering underlies the modern production of "bacterial" interferon.
The influence of genetic engineering on the technology of those medicinal substances that have long been created using biological technology has increased. Back in the 40s and 50s. 20th century was created
biological industry for the production of antibiotics, which are the most effective part of the drug arsenal of modern medicine. However, in recent years there has been a significant increase in drug resistance of bacteria, especially to antibiotics. The reason lies in the wide distribution in the microbial world of plasmids that determine the drug resistance of bacteria. That is why many previously famous antibiotics have lost their former effectiveness. So far, the only way to overcome bacterial resistance to antibiotics is to search for new antibiotics. According to experts, about 300 new antibiotics are created annually in the world. However, most of them are either ineffective or toxic. Only a few antibiotics are introduced into practice every year, which makes it necessary not only to maintain, but also to increase the capacity of the antibiotic industry on the basis of genetic engineering developments.
The main tasks of genetic engineering in those technologies of medicinal substances in which microorganisms are drug producers are determined by the need for genetic engineering reconstruction of the latter in order to increase their activity. At the same
At the same time, the idea of creating drugs in the form of small molecules began to be realized, which contributes to their greater effectiveness.
Immune biotechnology is primarily associated with the production of new generation vaccines for the prevention of infectious diseases in humans and animals. The first commercial products created with the help of genetic engineering were vaccines against human hepatitis, animal foot-and-mouth disease, and some others. An extremely important direction in this area is associated with the production of monoclonal antibodies, reagents necessary for the diagnosis of pathogens, as well as for the purification of hormones, vitamins, and proteins of various nature (enzymes, toxins, etc.).
Of considerable practical interest is the method of obtaining artificial hemoglobin by introducing hemoglobin genes into tobacco plants, where α- and β-globin chains are produced under the control of these genes, which are combined into hemoglobin. The hemoglobin synthesized in the cells of tobacco plants is fully functional (binds oxygen). Cell engineering as applied to humans is associated not only with the solution of fundamental problems of human biology, but also with overcoming, above all, female infertility. Since the frequency of positive cases of implantation in the uterus of women of embryos obtained in vitro, is small, then obtaining monozygotic twin embryos in vitro is also important, as the possibility of re-implantation increases due to "reserve" embryos. Of particular interest are the prospects for using stem cells as a source of cell and tissue replacement in the treatment of diseases such as diabetes, spinal cord injury, heart pain, osteoarthritis, and Parkinson's disease. But to realize these prospects, an in-depth study of the biology of stem cells is needed.
In the use of genetic engineering in relation to the problems of medicine, the task of developing genetic engineering methods for the radical treatment of hereditary diseases, which, unfortunately, are not yet treatable by existing methods, has acquired particular importance. The content of this task is to develop ways to correct (normalize) mutations that result in hereditary diseases, and to ensure the transmission of "corrections" by inheritance. It is believed that the successful development of genetically engineered methods for the treatment of hereditary diseases will be
contribute to the data on the human genome obtained as a result of the implementation of the international scientific program "Human Genome".
ENVIRONMENTAL PROBLEMS OF GENETIC ENGINEERING
Raising biotechnology to a new level, genetic engineering has also found application in the development of methods for determining and eliminating environmental pollution. In particular, bacterial strains have been constructed that are a kind of indicators of the mutagenic activity of chemical contaminants. On the other hand, bacterial strains containing plasmids have been genetically engineered to control the synthesis of enzymes capable of destroying many chemical compounds that pollute the environment. In particular, some plasmid-containing bacteria are capable of decomposing oil and oil products that have entered the environment as a result of various accidents or other unfavorable causes to harmless compounds.
However, genetic engineering is the transformation of genetic material, which does not exist in nature. Consequently, genetic engineering products are absolutely new products that do not exist in nature. Therefore, due to the unknown nature of its products, it itself poses a danger both to nature and the environment, as well as to personnel working in laboratories that use genetic engineering methods or work with structures created in the course of genetic engineering work.
Since the possibilities of gene cloning are endless, even at the very beginning of these studies, questions arose among scientists about the nature of the created organisms. At the same time, there were suggestions about a number of undesirable consequences of this methodology, and these suggestions also found support among the general public. In particular, disagreements arose about the properties of bacteria that received animal genes in genetic engineering experiments. For example, do bacteria retain E. coli their species affiliation due to the content of animal genes introduced into them (for example, the insulin gene) or should they be considered a new species? Further, how durable such bacteria are, in what ecological niches they can
exist? But the most important thing was the emergence of fears that during the production and manipulation of recombinant DNA molecules, genetic structures with properties that are unforeseen and dangerous for human health, for the historically established ecological balance can be created. At the same time, calls began for a moratorium on genetic engineering. These appeals caused an international outcry and led to an international conference held in 1975 in the USA, at which the possible consequences of research in this area were widely discussed. Then, in countries where genetic engineering began to develop, rules were developed for working with recombinant DNA molecules. These rules are aimed at preventing the products of the activities of genetic engineering laboratories from entering the habitat.
Another aspect of the undesirable consequences of genetic engineering work is related to the health hazard of personnel working in laboratories where genetic engineering methods are used, since such laboratories use phenol, ethidium bromide, UV radiation, which are harmful factors for health. In addition, in these laboratories there is the possibility of contamination with bacteria containing recombinant DNA molecules that control undesirable properties, such as drug resistance of bacteria. These and other points determine the need to improve the level of safety in genetic engineering work.
Finally, the problem of the danger of genetically modified products (genetically modified tomatoes, potatoes, corn, soybeans), as well as such products as bread, pastas, sweets, ice cream, cheese, vegetable oil, meat products, which in a number countries, especially in the United States, have become widespread. For 12,000 years of agriculture, humans have been using natural products. Therefore, it is assumed that with genetically modified food, new toxins, allergens, bacteria, carcinogens will enter the human body, which will lead to completely new diseases of future generations. This raises the question of a truly scientific assessment of genetically modified food.
ISSUES FOR DISCUSSION
1. What is meant by genetic, cellular and genetic engineering? Is there a difference between these concepts and molecular cloning?
2. What is the progressive nature of genetic engineering compared to other methods used in biology?
3. List the main "tools" of genetic engineering.
4. What are restriction enzymes, what are their properties and their role in genetic engineering?
5. Do all restrictases form "sticky" ends of the studied DNA and does the structure of "sticky" ends depend on the type of restrictase?
6. Define genetic vectors. Are there natural vectors?
7. How are genetic vectors obtained in the laboratory? What biological objects are the source material for obtaining vectors?
8. What is the maximum length of DNA nitrogenous base sequences that can still be included in a genetic vector? Do vectors differ in "power"?
9. Describe the properties of DNA ligase and determine its role in genetic engineering.
10. How is a cloned DNA segment (gene) linked to a genetic vector?
11. What is the frequency of introducing recombinant DNA molecules into bacterial cells?
12. On what principle is the selection of bacterial cells containing recombinant DNA molecules based? Give one example of such selection.
14. Many strains of bacteria have the same enzymes that provide almost the same metabolism. Meanwhile, the nucleotide specificity of bacterial restriction-modification systems is different. Can you explain this phenomenon?
15. Why can't DNA sequences representing restriction enzyme recognition sites contain more than eight base pairs?
16. How many times will the HHCC sequence recognized by the Hae III restriction enzyme occur in a 50,000 bp DNA segment with 30, 50, and 70 percent HC content?
17. Restriction enzymes Bam HI and Bgl I melt the G GATCC and T GATCA sequences, respectively. Can DNA fragments produced by Bgl I restriction be included in the Bam HI site? If yes, why? If the plasmid (vector) used contains one Bgl I restriction site, then on what nutrient medium can bacteria be selected, this plasmid?
18. Calculate the frequency of bacterial transformation per DNA molecule if 5-10 5 transformants are formed per 5000 plasmid base pairs?
19. Is it possible to clone the 0-point of DNA replication E. coli and if so, how?
20. Is it possible to determine how many DNA molecules are needed to transform one cell E. coli?
21. Is it possible to determine the splicing site on mRNA using polymerase chain reaction?
22. How can the polymerase chain reaction be used to introduce the desired restriction site at the site of interest on the DNA fragment to be cloned?
23. Name the methods of cell engineering as applied to animals. What is the economic value of animals produced by these methods?
24. Define the terms "transgenic plants" and "transgenic animals". Do transgenic organisms retain their species or can they be considered organisms of new species?
25. What are hybridomas and monoclonal antibodies? How are they received?
26. Is cell engineering applicable to humans?
27. Suppose that the injection of foreign DNA into a mouse egg and the implantation of the egg fertilized in this way into the body of a mouse ended in her pregnancy and the birth of mice containing copies of the injected DNA in the genome. However, the mice turned out to be mosaics; some of their cells contain copies of the injected DNA, while others lack this DNA. Can you explain the nature of this phenomenon?
28. Do you consider food prepared from genetically modified foods to be genetically hazardous?
29. Is scientific review of genetically modified foods necessary?
Cognition is determined by what we affirm as Truth.
P.A. Florensky, 1923
When applied to humans, genetic engineering could be used to treat hereditary diseases. However, technically, there is a significant difference between treating the patient himself and changing the genome of his descendants.
The task of changing the genome of an adult is somewhat more difficult than breeding new genetically engineered breeds of animals, since in this case it is necessary to change the genome of numerous cells of an already formed organism, and not just one embryonic egg. For this, it is proposed to use viral particles as a vector. Viral particles are able to penetrate into a significant percentage of adult cells, embedding their hereditary information into them; possible controlled reproduction of viral particles in the body. At the same time, to reduce side effects, scientists are trying to avoid the introduction of genetically engineered DNA into the cells of the genital organs, thereby avoiding exposure to the future descendants of the patient. It is also worth noting the significant criticism of this technology in the media: the development of genetically engineered viruses is perceived by many as a threat to all of humanity.
With the help of gene therapy in the future, it is possible to change the human genome. Currently, effective methods for modifying the human genome are under development and testing in primates. For a long time, the genetic engineering of monkeys faced serious difficulties, but in 2009 the experiments were crowned with success: a publication appeared in the journal Nature about the successful use of genetically engineered viral vectors to cure an adult male monkey from color blindness. In the same year, the first genetically modified primate (grown from a modified egg) gave offspring - the common marmoset.
Albeit on a small scale, genetic engineering is already being used to give women with some types of infertility a chance to get pregnant. To do this, use the eggs of a healthy woman. The child as a result inherits the genotype from one father and two mothers.
However, the possibility of introducing more significant changes in the human genome faces a number of serious ethical problems.
_____________________________________________________________________________________________
Genetic engineering (genetic engineering)
This is a set of techniques, methods and technologies for obtaining recombinant RNA and DNA, isolating genes from an organism (cells), manipulating genes and introducing them into other organisms.
Genetic engineering is not a science in the broadest sense, but is a tool biotechnology using the methods of such biological sciences as molecular and cellular biology, cytology, genetics, microbiology, virology.
An important component of biotechnology is genetic engineering. Born in the early 70s, she has achieved great success today. Genetic engineering techniques transform bacterial, yeast and mammalian cells into "factories" for the large-scale production of any protein. This makes it possible to analyze in detail the structure and functions of proteins and use them as medicines.
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 the cost was very high. To obtain 100 g of crystalline insulin, 800-1000 kg of pancreas are 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 at Genentech made the first 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 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 have side effects like animal insulin, and does not differ from it in biological activity. Subsequently, proinsulin was synthesized in E. coli cells, for which a DNA copy was synthesized on the RNA template using reverse transcriptase. After purification of the resulting proinsulin, it was split and native insulin was obtained, 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. The lack 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 by 6 cm. final pharmaceutical product. Thus, the amounts of hormone available were limited, moreover, the hormone produced by this method was heterogeneous and could contain slowly developing viruses. The company "Genentec" in 1980 developed a technology for the production of somatotropin using bacteria, which was devoid of these shortcomings. In 1982, human growth hormone was obtained in the culture of E. coli and animal cells at the Pasteur Institute in France, and since 1984 industrial production of insulin has begun 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 by similar methods.
The production of highly specific DNA probes is based on the technology of recombinant DNA, with the help of which they study gene expression in tissues, the localization of genes in chromosomes, and identify genes that have related functions (for example, in humans and chickens). 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. With this approach, a protein is isolated from the cell, the gene of 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 that carries it and its descendants will synthesize the altered protein. In this way, defective genes can be corrected and hereditary diseases treated.
If the hybrid DNA is introduced into a fertilized egg, transgenic organisms can be obtained that express the mutated gene and pass it on to offspring. The 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, as well as animal breeds with traits useful for humans, have been created. 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.
Now it is even difficult to predict all the opportunities that will be realized in the next few decades.
Genetic engineering is a field of biotechnology that includes the actions of rearranging genotypes. Even today, genetic engineering makes it possible to turn individual genes on and off, thus controlling the activity of organisms, and also to transfer genetic instructions from one organism to another, including organisms of another species. As geneticists learn more and more about the work of genes and proteins, it becomes more and more real to be able to arbitrarily program the genotype (primarily human), easily achieving any results: such as resistance to radiation, the ability to live under water, the ability to to the regeneration of damaged organs and even immortality.
Encyclopedic YouTube
-
1 / 5
Genetic engineering serves to obtain the desired qualities of a modified or genetically modified organism. Unlike traditional breeding, during which the genotype is only indirectly changed, genetic engineering allows you to directly interfere with the genetic apparatus, using the technique of molecular cloning. Examples of applications of genetic engineering are the production of new genetically modified varieties of crops, the production of human insulin using genetically modified bacteria, the production of erythropoietin in cell culture, or new breeds of experimental mice for scientific research.
The basis of the microbiological, biosynthetic industry is the bacterial cell. The cells necessary for industrial production are selected according to certain criteria, the most important of which is the ability to produce, synthesize, in the maximum possible quantities, a certain compound - an amino acid or an antibiotic, a steroid hormone or an organic acid. Sometimes it is necessary to have a microorganism that can, for example, use oil or wastewater as “food” and process them into biomass or even protein quite suitable for feed additives. Sometimes organisms are needed that can grow at elevated temperatures or in the presence of substances that are unquestionably lethal to other types of microorganisms.
The task of obtaining such industrial strains is very important; for their modification and selection, numerous methods of active influence on the cell have been developed - from treatment with potent poisons to radioactive irradiation. The purpose of these techniques is the same - to achieve a change in the hereditary, genetic apparatus of the cell. Their result is the production of numerous mutant microbes, from hundreds and thousands of which scientists then try to select the most suitable for a particular purpose. The creation of techniques for chemical or radiation mutagenesis was an outstanding achievement in biology and is widely used in modern biotechnology.
But their capabilities are limited by the nature of the microorganisms themselves. They are not able to synthesize a number of valuable substances that accumulate in plants, primarily medicinal and essential oil. They cannot synthesize substances that are very important for the life of animals and humans, a number of enzymes, peptide hormones, immune proteins, interferons, and many more simply arranged compounds that are synthesized in animals and humans. Of course, the possibilities of microorganisms are far from being exhausted. Of the abundance of microorganisms, only a tiny fraction has been used by science, and especially by industry. For the purposes of microorganism selection, of great interest are, for example, anaerobic bacteria that can live in the absence of oxygen, phototrophs that use light energy like plants, chemoautotrophs, thermophilic bacteria that can live at a temperature, as it was recently discovered, of about 110 ° C, etc.
And yet the limitations of "natural material" are obvious. They tried and are trying to circumvent the restrictions with the help of cell cultures and tissues of plants and animals. This is a very important and promising way, which is also implemented in biotechnology. Over the past few decades, scientists have developed methods by which single cells of a plant or animal tissue can be made to grow and multiply separately from the body, like bacterial cells. This was an important achievement - the resulting cell cultures are used for experiments and for the industrial production of certain substances that cannot be obtained using bacterial cultures.
Another direction of research is the removal from DNA of genes that are unnecessary for coding proteins and the functioning of organisms and the creation of artificial organisms based on such DNA with a "truncated set" of genes. This makes it possible to sharply increase the resistance of modified organisms to viruses.
History of development and achieved level of technology
In the second half of the 20th century, several important discoveries and inventions were made that underlie genetic engineering. Many years of attempts to "read" the biological information that is "recorded" in the genes have been successfully completed. This work was started by the English scientist Frederick Senger and the American scientist Walter Gilbert (Nobel Prize in Chemistry in 1980). As you know, genes contain information-instruction for the synthesis of RNA molecules and proteins in the body, including enzymes. In order to force a cell to synthesize new, unusual substances for it, it is necessary that the corresponding sets of enzymes be synthesized in it. And for this it is necessary either to purposefully change the genes in it, or to introduce new, previously absent genes into it. Changes in genes in living cells are mutations. They occur under the influence of, for example, mutagens - chemical poisons or radiation. But such changes cannot be controlled or directed. Therefore, scientists have concentrated their efforts on trying to develop methods for introducing into the cell new, very specific genes that a person needs.
The main stages of solving the genetic engineering problem are as follows:
- Obtaining an isolated gene.
- Introduction of a gene into a vector for transfer into an organism.
- Transfer of a vector with a gene into a modified organism.
- Transformation of body cells.
- Selection of genetically modified organisms ( GMO) and eliminating those that were not successfully modified.
The process of gene synthesis is currently very well developed and even largely automated. There are special devices equipped with computers, in the memory of which programs for the synthesis of various nucleotide sequences are stored. Such an apparatus synthesizes DNA segments up to 100-120 nitrogenous bases in length (oligonucleotides). A technique has become widespread that allows the use of polymerase chain reaction for DNA synthesis, including mutant DNA. A thermostable enzyme, DNA polymerase, is used in it for template synthesis of DNA, which is used as a seed for artificially synthesized pieces of nucleic acid - oligonucleotides. The reverse transcriptase enzyme makes it possible to synthesize DNA using such primers (primers) on a matrix of RNA isolated from cells. DNA synthesized in this way is called complementary (RNA) or cDNA. An isolated, "chemically pure" gene can also be obtained from a phage library. This is the name of a bacteriophage preparation, in whose genome random fragments from the genome or cDNA are inserted, reproduced by the phage along with all its DNA.
The technique of introducing genes into bacteria was developed after Frederick Griffith discovered the phenomenon of bacterial transformation. This phenomenon is based on a primitive sexual process, which in bacteria is accompanied by the exchange of small fragments of non-chromosomal DNA, plasmids. Plasmid technologies formed the basis for the introduction of artificial genes into bacterial cells.
Significant difficulties were associated with the introduction of a ready-made gene into the hereditary apparatus of plant and animal cells. However, in nature, there are cases when foreign DNA (of a virus or a bacteriophage) is included in the genetic apparatus of a cell and, with the help of its metabolic mechanisms, begins to synthesize “its own” protein. Scientists studied the features of the introduction of foreign DNA and used it as a principle for introducing genetic material into a cell. This process is called transfection.
If unicellular organisms or cultures of multicellular cells are modified, then cloning begins at this stage, that is, the selection of those organisms and their descendants (clones) that have undergone modification. When the task is to obtain multicellular organisms, then cells with a changed genotype are used for vegetative propagation of plants or injected into the blastocysts of a surrogate mother when it comes to animals. As a result, cubs with a changed or unchanged genotype are born, among which only those that show the expected changes are selected and crossed with each other.
Application in scientific research
Albeit on a small scale, genetic engineering is already being used to give women with some types of infertility a chance to get pregnant. To do this, use the eggs of a healthy woman. The child as a result inherits the genotype from one father and two mothers.
However, the possibility of making more significant changes to the human genome faces a number of serious ethical problems. In 2016, a group of scientists in the United States received approval for clinical trials of a cancer treatment method using the patient's own immune cells, subjected to gene modification using CRISPR / Cas9 technology.
Cell engineering
Cellular engineering is based on the cultivation of plant and animal cells and tissues capable of producing substances necessary for humans outside the body. This method is used for clonal (asexual) propagation of valuable plant forms; to obtain hybrid cells that combine the properties of, for example, blood lymphocytes and tumor cells, which allows you to quickly obtain antibodies.
Genetic engineering in Russia
It is noted that after the introduction of state registration of GMOs, the activity of some public organizations and individual deputies of the State Duma, who are trying to prevent the introduction of innovative biotechnologies into Russian agriculture, has noticeably increased. More than 350 Russian scientists signed an open letter from the Society of Scientists in Support of the Development of Genetic Engineering in the Russian Federation. The open letter notes that a ban on GMOs in Russia will not only harm healthy competition in the agricultural market, but will lead to a significant lag in food production technologies, increase dependence on food imports, and undermine the prestige of Russia as a state in which the course for innovative development has been officially announced [ the significance of the fact? ] .
see also
Notes
- Alexander Panchin Beating God // Popular Mechanics . - 2017. - No. 3. - S. 32-35. - URL: http://www.popmech.ru/magazine/2017/173-issue/
- In vivo genome editing using a high-efficiency TALEN system(English) . nature. Retrieved 10 January 2017.
- Elements - science news: monkeys cured of color blindness with gene therapy (indefinite) (September 18, 2009). Retrieved 10 January 2017.
