Lecture #5
Subject: Biotechnology and genetic engineering.
Questions: 1. The concept of biotechnology
2. Genetic engineering and its methods.
1. The concept of biotechnology
Modern biotechnology occupies a leading position in the system of biological, medical, veterinary and zootechnical research, is a new form of industrial technology, which is based on biological objects - animals, plants and microorganisms.
The main goal and objectives of biotechnology are aimed at developing methods and techniques that allow obtaining biologically active compounds (enzymes, hormones, vaccines), as well as constructing molecules of new substances and creating new forms of organisms that are not found in nature (chimeric molecules, animals).
In animal husbandry, various biotechnological methods (genetic and cell engineering) are widely used, with the help of which it is possible to speed up the selection process to create new highly productive breeds of agricultural crops. animals.
In biotechnology, two terms are used that differ in semantic content: "Gene engineering" - as a method of studying and influencing processes occurring at the level of molecules and genes, and the term "Genetic engineering" - as a set of methods carried out more broadly on cells and organisms generally.
In principle, both terms are synonymous and imply methods that provide alteration and reconstruction of genetic material, i.e. the formation of a new heredity.
The use of the achievements of genetic engineering is mainly in the following areas:
study of the organization of the genetic apparatus of higher organisms;
the use of microorganisms as producers of economically useful substances;
constructing new organisms by transplanting foreign genes, i.e. obtaining transgenic animals.
increasing the efficiency and speeding up the selection process;
increase in the reproduction rate of females;
preservation of valuable, small populations of the gene pool of endangered breeds;
obtaining offspring from barren, but genetically valuable animals;
increasing the resistance of animals to diseases;
obtaining monozygotic twins of one specific sex;
obtaining chimeras developing from embryos of 5-6 days of age of different animals (breeds, species) and combined into one;
increasing the fertility of cows by transplanting half of the embryo into both uterine horns.
Historically, biotechnology arose on the basis of traditional microbiological (mostly fermentation) industries. Many such "technologies" were unconsciously used in ancient times in the production of wine, beer, bread, sour-milk and fermented products.
With the help of biotechnology, dozens of expensive biologically active substances are currently obtained, including hormones, enzymes, vitamins, antibiotics, some drugs, such as insulin, interferon, and others.
For reference: insulin is a protein that regulates blood sugar; interferon is a protein that protects still unaffected cells from viruses (influenza).
However, before the advent of genetic engineering, interferon could only be obtained in trace amounts from leukocytes (white blood cells).
To obtain 1 gram of interferon, you need to process the blood from 90 thousand donors.
Biotechnological developments are intensively used in the creation of non-waste production processes in the processing of raw materials, water purification from oil, sewage, in the fight against agricultural pests. crops, obtaining fodder and food protein, biogas, etc.
So, with the help of microbes, about 1 ton of yeast containing 600 kg of protein is obtained from 1 ton of oil.
And one more thing: with reproduction, 1 bacterium (under optimal conditions for food, environment, and other factors) after 44 hours would be able to form such offspring, the mass of which corresponded to the mass of our planet (about 6,000,000,000,000,000,000,000 tons).
Biotechnological methods, 6000 years ago, were used by the peoples of Mesopotamia when making a heady drink, i.e. beer of those times.
The ancient Egyptians knew how to brew beer using yeast, sugar and fermentation. The Romans and Greeks used grape juice to make wine.
Based on the above, to the question of what is biotechnology, we can answer that it is the science of using living organisms and biological processes in production.
In connection with the above, the history of the emergence and development of biotechnology can be divided into three stages.
The first stage is the birth of biotechnology. For many hundreds of years, people, having no scientific understanding of microbiology, biochemistry and other sciences, have developed and practically successfully used biotechnology methods in bread-baking, cheese-making, wine-making, the manufacture of fermented milk products, i.e. ancient branches of economic activity.
The second stage (XIX century) is the formation of biotechnology as a science. The beginning of the rapid development of biotechnological sciences: genetics, microbiology, biochemistry, virology, physiology, embryology, etc.
The third stage (mid-70s of the XX century) is the development of biotechnology in various directions using the methods of genetic and cell engineering.
The first biotechnological methods in animal husbandry were artificial insemination of animals and forage ensiling.
For the first time in Russia in 1887 V.I. Shvedov transplanted crushing fertilized eggs - rat zygotes.
The history of bovine embryo transplantation begins in 1950, when O. Willem (USA) transplanted a fertilized egg from one heifer into another and received a live calf.
Of the European countries that began to use embryo transplantation as a method that accelerates the selection process and increases its efficiency, it is necessary to note France, Great Britain, Denmark, Germany, Italy, Belgium, and Slovakia.
The current stage in the development of biotechnology is associated with the discovery of new patterns in the life processes of organisms at the molecular level.
The development of biotechnology has led to the creation of industrial production for the production of various biological preparations for use in medicine, veterinary medicine, and the food industry.
Every year, the number of technologies, methods and preparations designed to increase animal productivity and product quality is increasing. There are more than 450 biotech companies around the world producing drugs to maintain the health of animals and increase their productivity.
Directions of biotechnology
One of the most promising areas is embryo cloning, i.e. obtaining the maximum number of offspring from highly productive animals.
For this, a method has been developed for creating identical (clones) embryos by introducing the nucleus of a cell of an embryo of a high-class animal into an unfertilized egg with a previously removed nucleus, of little value in terms of breeding; division of embryos into two, four, six and eight parts.
Single-cell "synthetic" embryos have learned to grow up to the 8-, 16- and even 32-cell stage in the laboratory. Therefore, they can not only be implanted in cows or frozen for storage, but also used for subsequent cloning. Thus, an unlimited number of embryos can be obtained in vitro, excluding the procedure for taking them from highly productive animals. Theoretically, from one embryo of cattle, you can have thousands of animals.
Another achievement of biotechnological research in the field of animal husbandry, which is of practical importance, is the method of obtaining transgenic animals with a foreign gene inserted into their genome. With its help, fast-growing animals with high milk production, disease resistance, etc. can be obtained in a short period.
Obtaining even one animal with an inherited gene transplant is considered a great achievement. Such an animal is considered as the basis for creating a new line.
Today, biotechnology is used to solve many practical issues to improve the efficiency of health care, increase the country's food resources and provide various industries with raw materials, create and use cost-effective renewable energy sources and waste-free industries, reduce harmful anthropological impacts on the environment and in other industries.
Currently, in the most developed countries, enterprises have been created and continue to be created that, using biotechnology, produce feed and feed additives, food products, medicines, carry out embryo transplantation and solve other economic problems.
It is believed that the further progress of mankind will not only largely depend on the development of biotechnology, but simply will not be able to do without it, since there are no other scientifically sound proposals to provide, first of all, food for the growing population of the Earth.
The most promising areas in biotechnology are productions associated with non-traditional production at biofactories, in the required quantities of protein, essential amino acids, medicines, biogas and solar energy conversion.
2. Genetic engineering and its methods
Modern genetic engineering uses a complex of various methods and technologies at the level of molecules, cellular elements (chromosomes, nuclei), somatic and germ cells, on an organism located on different stages ontogeny.
Interferon synthesis process by chemical means from the blood of animals is complex and lengthy. Therefore, we used microorganisms (E. Coli), obtained by genetic engineering, capable of producing human interferons, which activate processes that affect antiviral resistance.
Insulin, human growth hormone, interferon were obtained by genetic engineering methods in industrial conditions. Methods for the synthesis of albumin, various vaccines, certain enzymes, and growth hormone are being developed. animals.
On the basis of genetic engineering, gene therapy is being created, which makes it possible to correct hereditary defects by introducing full-fledged genes into the body. Giant mice were obtained in this way. The gene for growth hormone has been “embedded” into their genome.
3. Cellular and embryonic engineering.
Cellular engineering. Cell engineering is understood as a method of constructing a new type of cells based on their cultivation, hybridization and reconstruction.
One of the important areas of cell engineering is the hybridization of somatic cells.
Its essence lies in the connection of cells with chromosome sets of very distant species.
Somatic hybridization uses the ability of cells in culture to unite into one and form a nucleus containing chromosomes. different genomes. This is done using the Sendai virus.
At present, hybrid cell cultures of dozens of distant species have been obtained (mouse x chicken; mouse x monkey; rabbit x monkey; soybean x pea; soybean x corn, etc.).
It turned out to be possible to combine in one cell even such distant forms as chicken x yeast, etc.
However, interspecific incompatibility remains the law in somatic hybridization.
Over time, in a hybrid culture, there is a division into cells of both types, which do not contain chromosomes of the second type.
This circumstance turned out to be extremely valuable for studying the localization and nature of the action of certain genes.
embryonic engineering. This area includes embryo transfer. Biotechnology in the reproduction and selection of cattle is of particular importance. Cattle are monocytic mammals. At best, one calf per year is produced from each cow, while the ovary contains hundreds of thousands of immature germ cells - oocytes, representing a huge genetic reserve.
The cardinal solution to the problem of accelerated reproduction of livestock is to switch to non-traditional ways to increase fertility. In the future, biotechnology is considered as the basis for the accelerated reproduction of highly productive animals and entire populations.
The methods of biotechnology used in the practice of reproduction include artificial insemination, deep freezing and long-term storage of bull semen, induction of estrus and its synchronization, regulation of calving time.
Recently, along with these traditional biotechnical methods, embryo transplantation has gained practical importance, which is considered as effective method biotechnologies of accelerated reproduction of highly valuable breeding animals.
Bovine embryo transplantation is a new biotechnical method of accelerated reproduction of highly productive animals, which significantly increases the role of breeding stock, is an integral part of the breeding program and is one of the ways to intensify the use of the genetic potential of record-breaking cows. Embryo transplantation is effective only when genetically valuable animals are used, tested for the quality of offspring and recognized as improvers.
Considering that embryos can be obtained from one donor 4-5 times a year, then already at the present stage of development of transplantation biotechnology, the real possibility of obtaining 20-25 calves annually from one record-setting cow is obvious. Using 20 record cows as embryo donors, within 2-3 years it is possible to create a highly productive dairy herd of 200-300 cows. In the traditional way, no more than 30 heifers and 30 bulls can be obtained from the same 20 cows during this period.
Cows, heifers, to which embryos are transplanted, are usually called recipients, and cows from which embryos are obtained are called donors. The effect of transplantation is largely determined by the choice of cows. The best cows or heifers are used as donors, and the worst cows or heifers are used as recipients.
The higher the difference in quality between the donor and the recipient, the more expedient is the use of a transplantation method based on the use of cows with record high productivity as donors. For insemination of donor cows, the semen of the best bulls, evaluated by the quality of the offspring, is used.
The most important method of embryo transplantation can be in the breeding and selection of sires of outstanding breeding value, since the possibility of selecting bulls from mothers with record high productivity increases.
Obtaining transplant calves from outstanding parents does not reduce the problem of their subsequent assessment of the quality of the offspring, but significantly increases the likelihood of selecting (by increasing the breeding differential of mothers) outstanding improvers for use in breeding plants and under conditions of large-scale breeding.
The use of the method of embryo transplantation puts all breeding work on a new intensive path for the development of breeds, providing an increase in productivity due to the production and widespread use of producers with high combinational ability.
Embryo transplantation is developing rapidly, and the method itself is used abroad for commercial purposes. More than 80 commercial embryo transfer centers have been established in the United States. In this country, where embryo transplantation is put on a solid technological basis, more than 100 thousand calves are received annually. Similar commercial organizations for embryo transplantation have been established in other developed countries. Western Europe. In the USSR, developments in embryo transplantation began in the mid-1970s.
The purpose of the transplant is to:
Creation of breeds, lines, families or specialized types of animals;
Consolidation or improvement of existing breeds, lines, families of animals;
Crossbreeding of breeds, lines, families and interspecific hybridization;
Regulation of multiple pregnancy of farm animals;
Conducting scientific research and training of specialists (as an educational process).
Donor selection and poliovulation.
The most important criterion at the first stages of selection of donor cows is their high breeding value, i.e. the ability to pass genes of high productivity to their offspring. The breeding value of the donor must be confirmed not only by the high productivity of the cow itself, but also by her relatives. The group of donors selected as mothers of future sires includes the best cows of breeding herds.
First, the breeding value of a donor cow is determined by milk productivity with a completed lactation of 305 days, the content of fat and protein in milk, the suitability of cows for machine milking, the strength of the constitution and exterior.
When selecting, donors are subject to general and special requirements. The general requirements include the following:
The animal must be clinically healthy;
The donor must be assessed by type nervous system, exterior and constitution;
The donor should be evaluated by reproductive qualities (development and physiological state of the genital organs, the length of the service period, the quality and viability of the offspring);
Each donor must have a veterinary certificate indicating their clinical condition.
Special requirements include requirements that contribute to the achievement of the ultimate goal of embryo transfer, while taking into account the following:
The donor must be a typical representative of the breed, line, family in terms of exterior, constitution and economically useful features;
The donor must be tribally assessed using biometric methods;
The characteristic economically useful traits of the donor should be assessed for the possibility of their phenotypic compatibility in the planned animal genotypes.
The high costs of obtaining calves by embryo transplantation necessitate the selection of such donors from whom a large number of embryos can be obtained regularly. Preference should be given to cows that have maintained a stable reproductive capacity for three calvings. From donor cows with good and stable reproductive abilities, embryos can be regularly obtained every 2 months.
If we proceed from the generally accepted position that one calf per year should be obtained from a cow, then the intercalving period should not exceed 365 days on average. Therefore, getting one calf from each cow in 365 days is the main indicator of her good reproductive ability.
To assess reproductive ability, you can use the index of reproductive ability IVS, which is determined by the formula IVS \u003d (n-1) 365 100 D, where n is the number of calves received, D is the number of days between the first and last calving. With a stable reproductive ability, the index should not exceed 100.
The duration of the embryonic period in cows is on average 285 days, therefore, the optimal service period should not exceed 80 days. During this period, the cow must be fertilized.
After the selection of donor cows, multiple ovulation (poliovulation) is started. This method was developed by the Soviet embryologist M.M. Zavadovsky and his staff. They proved that if gonadotropic hormones are introduced into the blood of a female, this leads to the stimulation of the maturation of an additional number of follicles. Pregnant mare serum (FFK) was used as a gonadotropic hormone.
An important link in the modern biotechnology of bovine embryo transplantation is the hormonal induction of superovulation in donor cows. Only those cows that respond positively to the introduction of hormones are transferred to the donor group.
To stimulate multiple ovulation, FFA gonadotropin is used in combination with prostaglandins and other biologically active substances. This method allows you to induce poliovulation in about 70% of cows. The optimal result of poliovulation is the release of 10-20 eggs from the ovary into the funnel of the oviduct. The average number of ovulations is about 10, and the fertilization of eggs reaches 80%.
However, only a small proportion of donors show a recurrent ovarian response after induction of poliovulation. In general, donor cows respond irregularly to repeated hormonal treatment, i.e. one time they react well, and the other time they react badly. Therefore, the number of ovulations and the yield of embryos are not stable.
Poliovulation is also influenced by factors such as the stage of lactation, stillbirth or difficult calving, the time of estrus, the dose of FFA, the month of calving, the breed, economic conditions, the live weight of the donor, stress, the level and quality of feeding, etc.
It was found that the lengthening of the lactation period contributes to better reaction cows for injected FFA. The optimal time to induce poliovulation in lactating black-and-white cows is from day 60 after calving.
To optimize poliovulation and obtain biologically valuable embryos, it is necessary to provide a complete feeding of the donor, balanced in all nutrients.
The optimal dose for FFA donor cows is 2500-3000 IU. When injected at this dose, an average of 9 ovulations per positively responding donor is obtained.
The highest effect of poliovulation is achieved with the introduction of FFA between 10-12 days of the estrous cycle (mid-luteal phase) and after the 2nd day of prostaglandin, which causes regression of the corpus luteum, estrus and ovulation. Within 48 hours of prostaglandin injection, 95% of donor cows poliovulate with all signs of estrus.
Multiple poliovulation is subject to great variability. Therefore, not all donor cows have the same predisposition to multiple ovulation. For effective multiple poliovulation requires careful selection of donors for a number of indicators.
Producer selection.
When selecting bulls, they are evaluated by karyotype in order to exclude chromosomal abnormalities. The offspring of the selected sires must be free of conformational defects.
In the vast majority of cases, the selection of producers and donors is carried out according to the plan of custom mating in accordance with the breeding program. Sperm of producers selected for insemination of donors should be characterized by the highest fertility, not less than 85-90%.
The main criterion for the reproductive ability of producers is the indicator of the fertilizing ability of their sperm. Thus, in order to assess the sire being checked (bull, boar, ram) by the fertilizing ability of sperm, a control mating is organized. To do this, three or four groups of cows (800-1000 heads) are selected from different herds.
If the fertilizing ability of the sperm of the tested bull is 60% or less, then such a bull is culled. In practice, this indicator is determined by the number (percentage) of cows fertilized from the first insemination. Fertility is determined by the absence of estrus within 60-90 days after insemination.
Artificial insemination methods make it possible to determine the fertilizing ability of sperm much earlier. Thus, the intensity of culling bulls in terms of sexual activity and sperm quality is 25-30%. A significant amount of sperm (20-30%) is rejected when evaluating freshly obtained ejaculates, 10-15% - with biological control of sperm 24 hours after freezing.
Insemination of donor cows.
The effectiveness of poliovulation is subsequently determined by the effectiveness of artificial insemination of donors. The results of numerous studies show that only 60-65% of the resulting embryos are suitable for transplantation into recipients. The remaining 35-40% are eggs or degenerated embryos.
For artificial insemination of donor cows, it is necessary to use the sperm of only outstanding sires, reliably assessed by the quality of the offspring.
The requirements for assessing the fertility of bull semen intended for the insemination of donor cows should be significantly higher than for the insemination of other cows. The fertilizing ability of the sperm of such bulls should be at least 70% with a high accuracy of its assessment.
To increase the fertility of donors and the release of embryos, along with the use of high-quality sperm, it is necessary to determine the timing of estrus for timely artificial insemination. Many signs of poliovulation indicate that only a short period is most favorable for effective fertilization and obtaining biologically valuable embryos.
There are different opinions of experts about the time and frequency of insemination of cows with hormonally induced estrus. As a rule, such cows are inseminated twice: the first time at the beginning of estrus and the second time after 12-24 hours.
In our country, donor cows are artificially inseminated twice a day with an interval of 10-12 hours, each time with two or three doses of frozen semen.
Three methods are used for artificial insemination of donor cows: visual(using a vaginal speculum); manocervical(insertion into the vagina of a gloved hand and a shortened pipette); rectocervical(with fixation of the cervix and control of the progress of the insemination pipette with a hand inserted into the rectum).
The highest efficiency of artificial insemination of donor cows is provided by the rectocervical method, which allows you to control the state of the donor's genital tract. The day on which the artificial insemination of the donor cow is carried out is considered the date of insemination.
Monoclonal antibodies are immunoglobulins synthesized by a single clone of cells.
Promising is the hybridization of cancer and normal cells, on the basis of which hybrids are obtained - hybridomas that produce monoclonal antibodies.
A hybridoma is understood to mean a hybrid cell obtained by fusion of an antibody-producing cell with a cancer cell, giving the hybridoma the ability to reproduce unrestrictedly when cultivated in vitro.
In this case, hybridomas inherit from a normal parent cell the ability to produce a valuable biological substance - an antibody, and from a cancer cell - the ability to unlimited growth and the formation of a monoclone.
An antibody is a protein synthesized by the body's immune (protective) system that binds specifically to an antigen.
As a result of a protective reaction to an antigen (foreign protein), a whole combination of various antibodies is formed, representing an indivisible mixture.
Therefore, it is impossible to isolate the desired antibody in pure form by traditional methods.
It is possible to obtain pure antibodies of a certain line if we isolate the cell producing the antibody we need and form a clone from it.
Anti-forming cells are not able to grow in a nutrient medium. Therefore, they must be fused (ie connected) with myeloma (cancer cells) cells capable of unlimited division in a nutrient medium and the production of monoclonal antibodies.
Methods of extraction of embryos and their evaluation.
The efficiency of the transplantation method is largely determined by the way the embryos are retrieved. There are different ways to retrieve embryos. The simplest is the slaughter of the donor. It was used in the early stages of the development of transplantation for demonstration as an educational practice and for scientific purposes. The time between the slaughter of the donor and the washing out of the embryos should not exceed 30-40 minutes, i.e. embryos must be obtained before the process of cellular digestion in the genitals begins. Currently, due to the loss of a genetically valuable donor cow, it is not used.
Surgical extraction of embryos was used in the 70s. Embryos are retrieved between 7-8 days after the first artificial insemination. With washout, an average of 5 embryos can be obtained from each donor. Three methods have been developed: extraction of embryos through an incision in the upper fornix of the vagina; laparotomy along the white line of the abdomen (under anesthesia of the donor); laparotomy in the region of the hungry fossa with the use of local anesthesia. The surgical method of extracting embryos is more laborious; highly qualified surgeon, operating room and sterile conditions are required. Therefore, it is used in rare cases, only for scientific purposes.
The catheter (non-surgical) method of embryo extraction practically does not cause any complications in the animal's body. The catheter method can successfully retrieve embryos in a livestock building. The effectiveness of the method is high, multiple. Using the catheter method, an average of 4.3 normal embryos are obtained from 86% of donor cows. On average, from washed eggs, up to 25% are unfertilized or degenerated. The embryos are washed out on the 7-8th day after insemination. For washing, Dulbecco's nutrient medium is used. The duration of the manipulation is 20-50 minutes.
To obtain embryos in this way, special catheters have been developed. The washing medium is injected into the horns of the uterus 5-8 times and removed from them with a syringe. Washing the uterine horns ensures the extraction of up to 76% of embryos from the number of ovulations. The main part of the embryos, more than 50%, is extracted in the first three or four washes at the morula or blastocyst stage with 32 or 64 blastomeres.
After washing the embryos, an antibiotic solution is injected into the uterus for the purpose of antisepsis.
Before transferring an embryo to a recipient, it is necessary to evaluate its quality and determine the method of transfer. Embryos are evaluated by various methods: morphological, intravital staining, cytological, etc.
It is believed that the degree of accuracy of the morphological method can be increased to 90% or more. Embryos from the oviduct enter the uterus on the 3rd-5th day, however, within 10-15%, they can also arrive after 6-8 days. Embryos that have not reached a certain stage of development at a specified time, as a rule, die. In this regard, the quality of embryos is assessed on the 7-8th day according to the degree of their development to the blastocyst.
In the morphological assessment, special attention is paid to the external shape of the zygote, the state of the pelucid zone, the number of blastomeres, the uniformity of crushing, the severity of the embryoblast and trophoblast, the clarity in the outline of the cells, the vacuolization of the cytoplasm - the clarification of its periphery, the destruction of the cytoplasm (churning into a lump), the integrity of the cell membrane and release of the cytoplasm.
On early stages crushing, special importance is attached to the configuration of blastomeres. The normal configuration provides intimate contact with the greatest number of cells with the least volume. Improper fragmentation of embryonic cells leads to disruption of the spatial arrangement of blastomeres, which leads to disruption in subsequent stages of development. Cow embryos are evaluated on the 7th-8th day after the first insemination under a microscope at 100-160-fold magnification.
In Russia, a 5-point school for assessing the quality of embryos has been adopted, taking into account: the integrity of the transparent membrane, the uniformity of crushing, the state of the cytoplasm, the transparency of the perivitelin space, and the correspondence to the stage of development. The most suitable for transplantation are embryos rated 4-5 points, which are in the late morula or blastocyst stage.
To improve the morphological assessment, fluorescent staining is additionally used, which makes it possible to distinguish live embryos from dead ones.
Evaluation of the quality of embryos by their staining method is based on the ability of dyes to stain the morphological structures of living and dead cells. Life-time coloring of embryos is carried out with non-toxic dyes.
The ideal embryo should be compact, spherical in shape, uniform in color, with cells of the same size, with a smooth, flat and evenly formed pellucid zone, without inclusions in the peritoneal space.
An important criterion for assessing the quality of embryos is the intensity of development stages. Embryos with delayed development are not used for transplantation, freezing, etc.
Degenerated unfertilized eggs, which can be detected during the extraction of embryos, are subject to culling. Embryos unsuitable for transplantation have a defective morula or blastocyst, the signs of which are defects in the transparent membrane, disintegration of blastomeres, different sizes of blastomeres, disruption of intercellular communication.
Short-term cultivation, cryopreservation and storage of embryos.
Short-term storage and cultivation (development) of embryos makes it possible to transport them to other farms. Currently, the method of short-term storage of embryos in vitro has become widespread. It has been established that cow embryos can continue their development up to certain stages at the animal's body temperature in special cultivated (nutrient) media and under certain atmospheric conditions.
After extraction and evaluation for viability, the embryos are transferred to nutrient media at a temperature of 37 0 C. Several nutrient media have been developed for short-term storage of embryos in vitro (95 hours). Most often, the following are used as nutrient media for culturing embryos: TS-199; Ham F-10; Needle, saline solutions of Dulbek, Brinster with various biological and synthetic additives. For supporting optimal conditions development of embryos use a gaseous environment containing 90% nitrogen, 5% oxygen and 5% carbon dioxide. Cultivation of embryos in test tubes or straws is a simpler method that allows the transport of embryos over long distances.
The second method of short-term storage of embryos is carried out at a temperature of 8-12 0 C, the duration is 3-4 days. The cooling rate is slow to avoid thermal shock. For this, synthetic media with various protein supplements are used: bovine serum albumin (BSA), blood serum of boars castrates (SCH), and normal serum of steers castrates (NSBK).
The third method of short-term storage of embryos takes place in vivo, i.e. in the genital organs of an intermediate recipient (rabbits, mice, etc.) for transportation over a long distance.
The method is based on the high tolerance (tolerance) of the mucous membrane of the female genital tract during estrus and hunting to foreign proteins. For this, in 1982. a special storage chamber for embryos was designed for recipients in abdominal cavity. In such a chamber, embryos are stored for 72 hours.
The efficiency of transplantation of bovine embryos is largely determined by the conditions of storage of zygotes. The most effective and promising method for preserving embryos is their deep freezing (cryopreservation) in liquid nitrogen at a temperature of -196 0 C. This method significantly expands the possibilities of transplantation and is a reliable biotechnological basis for animal breeding.
When storing frozen embryos (-196 0 C), there are a number of advantages that allow you to transfer embryos at any time, create a “bank” of embryos from high-value breeding animals, small and endangered breeds, and transport embryos at any time of the year.
For freezing of embryos, automatic program freezers UOP-12 are used. To protect embryos from destruction during freezing and thawing, special cryoprotective substances are used that easily penetrate into the cell - cryoprotectant glycerol.
Before freezing, the embryos are placed in a cryoprotectant with an increasing concentration of substances to balance the osmotic pressure. Exists fast way cryopreservation: cooling from +20 0 C to -6 0 C at a rate of 1 0 C/min. Subsequent cooling down to -35 0 C at a rate of 0.3 0 C/min. Next, transfer the embryo into liquid nitrogen.
Embryos are thawed at 25 or 37 0 C for 10-12 s. Then they are washed from the cryoprotectant and evaluated. Embryo survival should be at least 80%, pregnancy 55-60%, in this case, transplantation is zootechnically and economically viable.
Selection, preparation and transfer of embryos to recipients.
On average, 5-6 recipients are selected per donor, taking into account possible subsequent culling due to their unsuitability for reproduction. Recipient cows must be no older than 7 years old with no gynecological abnormalities, good breeding conditions and reproductive qualities. Recipient heifers must be 16-18 months of age with a live weight of 350-380 kg.
The results of embryo transfer in cows are high only if the day of ovulation for donors and recipients coincides in time, then the mucous membranes of the genital organs of donors and recipients are in identical physiological states.
To do this, group synchronization of sexual hunting is carried out. Differences in synchronization should not exceed 12 hours. Delay of estrus in recipients by 10-12 hours significantly reduces the percentage of survival of embryos, and advancing estrus by 12 hours does not affect the efficiency of graft engraftment.
With proper synchronization, it is possible to achieve 90% of the recipients' pregnancy, while the discrepancy in the manifestation of estrus between the donor and the recipient for more than 24 hours reduces the pregnancy to 50% and below.
Methods for transplanting embryos by recipients have been developed - surgical and non-surgical. In surgical methods of embryo transfer, laparotomy is used along the white line of the abdomen or in the iliac region. Laparotomy is performed under general anesthesia in the dorsal position of the animal. The length of the incision along the white line of the abdomen is 10 cm.
Until the 1970s, the extraction and transplantation of bovine embryos was mainly performed surgically. However, it requires a lot of money. Therefore, in the last 10-15 years, embryo transfer has been mainly carried out in a non-surgical way.
The main advantage of the non-surgical method of embryo transfer, in addition to the simplicity of great economy, is the possibility of multiple use of the recipient. Several methods have been developed, but they are all based on the same principle - the introduction of the embryo into the uterine horn through the cervix, as a result of which this method is called cervical. The catheter, in which the straw with the embryo is located, is carefully inserted to the cervix and, under rectal control, is passed through the cervical canal, deep into the uterine horn closer to its upper part, and the embryo is pushed along with the medium into the lumen of the uterine horn.
On the 60th day after embryo transfer, recipients are checked for pregnancy by rectal palpation. This method is classical and gives great accuracy.
The introduction of methods for transplanting embryos and increasing the multiplicity of cows necessitates determining the origin of calves. For this, groups of animals with their antigens are used. Identical blood types are possible only in identical twins. Determination and clarification of the origin of calves is necessary due to the fact that there may be errors in the conduct of breeding records, the use of semen from different bulls. Animal blood groups are determined in special laboratories with monospecific sera.
In vitro fertilization and development of embryos outside the body.
Currently, much attention is paid to studying the mechanism of in vitro fertilization of oocytes, or in vitro fertilization (i.e., outside the animal body), which makes it possible to more intensively use cows of high breeding value in reproduction, which will dramatically increase genetic progress in the population.
Currently, methods have been developed that allow isolating up to 200 oocytes from cow ovaries, cultivating them and fertilizing them in vitro. However, the yield of full-fledged embryos remains extremely low, so research continues to develop new and improve existing methods.
Fertilization of oocytes in vitro has been achieved in 20 species of mammals, incl. and in humans, in 1981 normal offspring were obtained. The process of fertilization of gametes takes place in vitro and under controlled conditions. The final evaluation of true in vitro fertilization of oocytes is the transplantation of the zygote into the recipient and the birth of a live animal.
Cultivation of oocytes in vitro.
In vitro fertilization is preceded by in vitro cultivation of oocytes.
Under the cultivation of oocytes in vitro is understood the process of maturation of immature oocytes in artificial nutrient media, in which immature oocytes undergo meiotic maturation up to the metaphase of the second division, i.e. to the stage of readiness for fertilization.
For the isolation of oocytes from follicles, as a rule, ovaries from slaughtered cows are used and, less often, ovaries extracted by surgery. After extraction, the best ovaries (2-6 mm in diameter) are selected, the rest are discarded. The most acceptable method of extracting oocytes from follicles is by cutting them with a blade. Under the control of stereo microscopes MBS-9 and MBS-10 selected oocytes with compact cumulus.
To evaluate oocytes by viability, several methods have been developed, the most widely used of which is morphological.
To the main morphological features characterizing the biological usefulness of oocytes include the structure of kumklus cells and the oocyte itself.
The 2-6 mm oocyte is surrounded by cumulus cells. A compact, multilayered cumulus that is tightly adjacent to the oocyte serves as a criterion for resistance to atretic changes in the follicle from which the oocyte was extracted.
Oocytes suitable for cultivation must meet the following requirements: round shape; ooplasm fine-grained, homogeneous, evenly filling the entire oocyte; transparent shell uniform in width, opalescent, rounded; cumulus compact, multi-layered, closely adjacent to the oocyte, homogeneous.
The viability of oocytes is determined using fluorescent dyes. Non-viable cells are stained after 7-10 min, while viable cells are not stained. Oocytes that respond necessary requirements put on cultivation.
Several methods for culturing oocytes have been developed. The main ones are: cultivation in closed vessels; in Petri dishes in a nutrient medium coated with a layer of vaseline oil. With any cultivation methods, the following is required: sterility at all stages of work; gas sterile; temperature 39 0 C at maximum humidity.
For the cultivation of mammalian oocytes, depending on the animal species, two types of culture media are used: simple and synthetic.
In all media with the indicated additives (LH, FSH, etc.), 80% of oocytes reach the stage of metaphase of the second division of maturation. Thus, during the maturation of oocytes in vitro, the first meiotic division is completely completed, and the second division of the maturation of most oocytes ends with the stage of metaphase II meiosis. The final completion of meiosis occurs after fertilization.
Changes in protein synthesis of oocytes are associated not with nuclear, but with cytoplasmic maturation, which is crucial for normal fertilization and early embryonic development up to implantation of the embryo into the recipient's uterine wall.
Sperm capacitation.
In order for sperm to fertilize an egg, changes must occur in them that characterize capacitation, i.e. their readiness for fertilization.
Sperm capacitation (maturation) is understood as a complex of physiological and physicochemical changes, as a result of which sperm acquire the ability to penetrate the zona pellucida, penetrate and fertilize the egg.
Under natural conditions, capacitation occurs during the passage of spermatozoa through the female genital tract, where they are separated from the seminal plasma. Capacitation can be carried out in vitro if the sperm cells are in certain culture and gas environments.
For the capacitation of spermatozoa of cattle, culture media have been developed: Krebs-Ringer, Tyrode, Brinster. The duration of sperm capacitation in these media is 8 hours.
For in vitro fertilization, deep-frozen sperm is used, packed in bags.
A particular problem is the objective assessment of sperm capacitation. The acrosomal reaction is used to prove capacitation. After the attachment of spermatozoa to the zona pellucida of the egg, an acrosomal reaction occurs.
The acrosome is a sperm organelle rich in various enzymes and located under the plasma membrane surrounding the sperm head. During the acrosomal reaction, enzymes are released that determine the fertilizing ability of sperm. Enzymes destroy the zona zona pellucida, which allows sperm to advance into the ooplasm of the oocyte. Of the many spermatozoa that have penetrated the zone of the oocyte pellucid, only one merges with the plasma membrane of the egg and fertilizes it. A zygote develops.
In vitro fertilization of mature oocytes in vitro is as follows. Bovine oocytes that have reached the stage of metaphase II maturation are fertilized with capacitated sperm. At a number of types of page - x. In animals, depending on the quality of in vitro matured gametes, the fertility rate is 50-70%. The main reason for the decrease in the ability of fertilized eggs to embryonic development in vitro is the imperfection of culture media for early embryos, as a result of which the development of embryos is blocked at the stage of 8-16 blastomeres.
Obtaining embryos from in vitro fertilized oocytes.
The ultimate goal of in vitro fertilization of in vitro matured oocytes is to obtain embryos suitable for transplantation. When cultivating early bovine embryos, in most cases, embryonic development is blocked at the stage of 8-16 cells, i.e. when under natural conditions the embryos pass from the oviduct to the uterus. Only a few embryos develop to the late morula and blastocyst stages suitable for transplantation.
Embryos are incubated in two ways: in the oviduct of a rabbit, sheep or cow and in culture media such as TC-199; HEM-F-10; MRM, saline solutions of Dulbecco, Bringster with various biological and synthetic additives.
The first control over the development of the embryo is carried out 24 hours after fertilization. Syngamy, i.e. the entry into close contact of the pronuclei, resulting in the final fusion of male and female gametes, is observed 19 hours after in vitro fertilization and the formation of a two-cell embryo after 22 hours.
In 1983, the first calf was born from a follicular oocyte matured in vitro after its in vitro fertilization. Despite significant positive results in in vitro fertilization of oocytes and obtaining embryos in vitro, many problems, such as improving the cultivation of oocytes in vitro, capacitation of spermatozoa, etc., remain unresolved.
Animal cloning
The term "clone" (shoot) was first used in 1903 by Weber (Germany) in relation to plants propagating vegetatively and meant that the daughter plants of the clone are genetically identical to the parent.
Cloning- obtaining offspring that are an exact genetic copy of the organism. The set of such descendants - copies originating from one organism, is called a clone. Organisms within each clone are characterized by the same phenotypic uniformity and identical genotype.
Methods for obtaining genocopies:
1. Transplantation of somatic cell nuclei into an enucleated egg;
2. Induction of parthenogenesis (androgenesis, gynogenesis), which allows the genotype or mother to father to be completely transferred to the descendants.
A differentiated somatic cell contains a complete set of genes given organism. The karyotype of such cells does not differ in any way from the karyotype of a fertilized egg (zygote).
In animals in somatic cells after their differentiation (7-8 days), a stable repression or inactivation of a part of the genome occurs, which limits the use of differentiated cell nuclei in cloning.
Cloning steps:
Extraction of nuclei (blastomeres in 8-16 cell embryos);
Separation of the recipient egg into nucleated and non-nuclear fragments (obtaining an enucleated egg);
Fusion of an enucleated egg with a nucleus (blastomeres) using an inactivated Sendai virus or an electric field;
The room of the reconstructed zygote is an ogar cylinder;
Cultivation of embryos in the oviducts of intermediate recipients up to the blastocyst stage (7-8 days);
Blastacyst transplantation to the final recipient.
Chimaira- a fire-breathing animal, a monster with a lion's head, a goat's body and a dragon's tail.
Chimera- a composite, composite animal, consisting of genetically heterogeneous cell populations, originating from more than one fertilized egg.
From a genetic point of view chimeras- this is the product of the union of 2 or earlier embryos, as a result of which they have a complex combined genotype.
Chimeras- hybrid animals, splitting occurs in the descendants, but there is no recombination of the genes of the original breeds or species, therefore chimeras retain the characteristics and properties of the original forms only in 1 generation.
Methods for obtaining chimeras
Aggregation method for creating chimeras
Developed by V. Tarkovsky (1961) and B. Mints (1962) upon receipt of chimeric mice.
Embryos are removed from the oviducts of females on the 4-5th day after fertilization (8-16 blastomeres), treated with the enzyme pronase in order to free them from the transparent membrane and bring them together with a glass microneedle or pushing the strings from a micropipette in a nutrient medium under a layer of paraffin oil on a heated table microscope (t 0 +37 0 С). Pooled embryos are cultured for 24-48 hours until aggregation is complete.
Injection method for creating chimeras
Developed by R. Gardner (1968).
In this case, embryos are used at the blastocyst stage (7-8 days). The embryo is held with a suction pipette attached to the manipulator, by piercing the transparent shell, a hole is made with 2 glass needles and stretched. A third needle is inserted into the formed gap and with its help the gap turns into a hole - a mold into which the inner cell mass of the donor embryo is injected with an injection pipette.
Obtaining transgenic animals.
Modern methods of selection of page - x. animals are based on the use of intraspecific genetic variability. As a rule, species are genetically isolated from each other, i.e. do not interbreed, because This is prevented by the so-called reproductive isolation mechanisms:
a) prezygotic - prevent the formation of zygotes;
b) postzygotic - a decrease in the viability and fertility of animals.
To overcome the biological boundaries of species and use interspecies genetic variability to create new forms of animals, it is possible with the help of gene transfer.
The transfer of foreign genes is understood as the transplantation outside the body of recombinant DNA molecules in the cell of another animal, regardless of species.
If a foreign gene has integrated into the genome of another animal, then such a gene is designated as a transgene, and the animals are called transgenic. The protein encoded by the transgene is called the transgenic product. If animals pass transgenes to their offspring, then transgenic lines are formed.
If the integration of a foreign gene has occurred in the cells of higher animals, then they become carriers of new hereditary properties and produce new substances for them.
Three methods are used to transfer genes to mammals:
Microinjection of recombinant DNA into the pronucleus of the zygote;
Use of retroviruses as vectors;
Injection of transformed embryonic stem cells into the embryo.
State educational institution Supreme
VlSU
Department of History and Religious Studies
Essay
on the topic of:
Genetic and cell engineering. Biotechnology.
Completed by: Shipilova E.V. Gr.ZYU-110
Checked by: Associate Professor of the Department of History and
Religious Studies Zubkov S.A.
Vladimir 2011
1. Introduction 3
2.Possibilities of genetic engineering . Biotechnology 5
3.1. Agriculture 9
3.2 Medicine and pharmaceuticals 11
4. Cloning 14
4.1 State of research on therapeutic
cloning in Russia 16
5. Issues 17
6. Conclusion 23
References 25
1. Introduction
Genetic engineering is a branch of research in molecular biology and genetics, the ultimate goal of which is to obtain, using laboratory methods, organisms with new, including those not found in nature, combinations of hereditary properties. Genetic engineering is based on the possibility of targeted manipulation of fragments due to the latest achievements in molecular biology and genetics. nucleic acids. These achievements include the establishment of the universality of the genetic code, that is, the fact that in all living organisms the inclusion of the same amino acids in a protein molecule is encoded by the same nucleotide sequences in the DNA chain; advances in genetic enzymology, which provided the researcher with a set of enzymes that make it possible to obtain individual genes or nucleic acid fragments in an isolated form, to carry out the in vitro synthesis of nucleic acid fragments, and to combine the obtained fragments into a single whole. Thus, changing the hereditary properties of an organism with the help of genetic engineering is reduced to constructing a new genetic material from various fragments, introducing this material into the recipient organism, creating conditions for its functioning and stable inheritance.
Genetic engineering arose in the beginning. 70s 20th century Genetic engineering is based on extracting a gene (coding the desired product) or a group of genes from the cells of an organism and combining them with special DNA molecules (so-called vectors) that can penetrate into the cells of another organism (mainly microorganisms) and multiply in them , i.e. creation of recombinant DNA molecules.
Recombinant (foreign) DNA introduces new genetic and physico-biochemical properties into the recipient organism. These properties include the synthesis of amino acids and proteins, hormones, enzymes, vitamins, etc.
The use of genetic engineering methods opens up the prospect of changing a number of properties of an organism: increasing productivity, resistance to diseases, increasing growth rate, improving product quality, etc. Animals that carry a recombinant (foreign) gene in their genome are commonly called transgenic, and a gene integrated into the genome recipient, transgenome. Thanks to the transfer of genes, new qualities arise in transgenic animals, and further selection makes it possible to fix them in the offspring and create transgenic lines.
Genetic engineering methods make it possible to create new plant genotypes faster than classical breeding methods, and it becomes possible to purposefully change the genotype - transformation.
Genetic transformation consists mainly in the transfer of foreign or modified genes into eukaryotic cells. In plant cells, the expression of genes transferred not only from other plants, but also from microorganisms and even animals is possible.
Obtaining plants with new properties from transformed cells (regeneration) is possible due to their topipotency property, i.e. the ability of individual cells in the process of implementing genetic information to develop into a whole organism.
2. Possibilities of genetic engineering. Biotechnology.
Currently, the pharmaceutical industry has gained a leading position in the world, which is reflected not only in the volume of industrial production, but also in the financial resources invested in this industry (according to economists, it entered the leading group in terms of the volume of purchase and sale of shares in the markets valuable papers). An important novelty was the fact that pharmaceutical companies have included in their sphere the breeding of new varieties of agricultural plants and animals, and spend tens of millions of dollars a year on this, they also mobilized the production of chemical substances for everyday life. Additives to products of the construction industry and so on. Already not tens of thousands, but perhaps several hundred thousand highly qualified specialists are employed in the research and industrial sectors of the pharmaceutical industry, and it is in these areas that interest in genomic and genetic engineering research is exceptionally high.
Obviously, therefore, any progress in plant biotechnology will depend on the development of genetic systems and tools that will allow more efficient management of transgenes. The situation is similar to that observed in the computer industry, where, in addition to increasing the amount of information processed and improving the computers themselves, we also need OS information management, such as microsoft "windows".
For the clean excision of transgenic DNA into the plant genome, homologous recombination systems borrowed from microbial genetics, such as the Cre-lox and Flp-frt systems, are increasingly being used. The future will obviously lie with controlled gene transfer from variety to variety, based on the use of pre-prepared plant material, which already contains in the required chromosomes the homology regions necessary for the homologous insertion of the transgene. In addition to integrative expression systems, autonomously replicating vectors will be tested. Of particular interest are plant artificial chromosomes, which theoretically do not impose any restrictions on the amount of theoretical information introduced.
Scientists are looking for genes that code for new useful traits. The situation in this area is changing radically, first of all, the existence of public databases that contain information about most genes, bacteria, yeast, humans and plants, and also due to the development of methods that allow the simultaneous analysis of the expression of a large number of genes with a very high throughput. The methods used in practice can be divided into two categories:
1. Methods that allow expression profiling: subtraction hybridization, electronic comparison of EST libraries, "gene chips" and so on. They allow you to establish a correlation between a particular phenotypic trait and the activity of specific genes. 2. Positional cloning consists in creating, through insertional mutagenesis, mutants with disturbances in the trait or property of interest to us, followed by cloning of the corresponding gene as such, which obviously contains a known sequence (insertion). The above methods do not imply any initial information about the genes that control this or that trait. The absence of a rational component in this case is a positive circumstance, since it is not limited by our current ideas about the nature and genetic control of a particular trait of interest to us.
Significant progress has been made in the practical field of creating new products for the medical industry and the treatment of human diseases.
The use of genetically engineered products in medicine.
| Natural products and scope of genetically engineered products | |
Anticoagulants | Tissue plasminogen activator (ATP) activates plasmin. An enzyme involved in the resorption of blood clots; effective in the treatment of patients with myocardial infarction. |
blood factors | Factor VIII accelerates the formation of clots; deficient in hemophiliacs. The use of genetically engineered factor VIII eliminates the risk associated with blood transfusion. |
| Factors stimulating the formation of colonies | Growth factors of the immune system that stimulate the formation of white blood cells. It is used to treat immunodeficiency and fight infections. |
erythropoietin | Stimulates the formation of red blood cells. Used to treat anemia in patients with renal insufficiency. |
| growth factors | Stimulate differentiation and growth various types cells. Used to speed up the healing of wounds. |
| human growth hormone | Used in the treatment of dwarfism. |
| human insulin | Used to treat diabetes |
Interferon | Prevents the reproduction of viruses. Also used to treat some forms of cancer. |
Leucins | Activate and stimulate the work of various types of leukocytes. Can be used for wound healing, HIV infection, cancer, |
Monoclonal nye antibodies | The highest specificity associated with antibodies is used for diagnostic purposes. are also used for targeted delivery of drugs, toxins, radioactive and isotopic compounds to cancerous tumors in cancer therapy, there are many other applications. |
| Superoxide dismutase | Prevents tissue damage by reactive hydroxy derivatives under conditions of short-term oxygen deficiency, especially during surgical operations when you need to suddenly restore blood flow. |
| Artificially produced vaccines (the first vaccine against hepatitis B was obtained) are better than conventional vaccines in many respects. |
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 mutant 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.
3. Directions of genetic engineering.
3. 1 Agriculture.
Genetic engineering directly in agriculture took place already in the late 1980s, when it was possible to successfully introduce new genes into dozens of plant and animal species - to create tobacco plants with luminous leaves, tomatoes that easily tolerate frosts, corn that is resistant to pesticides.
One of the important tasks of genetic engineering is to obtain plants resistant to viruses, since at present there are no other ways to combat viral infections of agricultural crops. The introduction of virus envelope protein genes into plant cells makes plants resistant to this virus. Currently, transgenic plants have been obtained that can withstand the effects of more than a dozen different viral infections.
Another important task of genetic engineering is related to the protection of plants from insect pests. The use of insecticides is not always effective due to their toxicity and the possibility of washing insecticides from plants with rainwater. In the genetic engineering laboratories of Belgium and the USA, work was successfully carried out to introduce into the plant cell the genes of the earthen bacterium Bacillus thuringiensis, which allow the synthesis of insecticides of bacterial origin. These genes were introduced into potato, tomato, and cotton cells, as a result of which transgenic potato and tomato plants became resistant to the Colorado potato beetle, and cotton plants were resistant to various insects, including the cotton bollworm. The use of genetic engineering in agriculture has reduced the use of insecticides by 40-60%. Genetic engineers have bred transgenic plants with an extended fruit ripening period. This makes it possible to remove such tomatoes from the bush red with the confidence that they will not overripe during transportation.
The list of plants to which genetic engineering methods have been successfully applied is growing. It includes apple, grapes, plums, cabbages, eggplants, cucumbers, wheat, rice, soybeans, rye and many other crops.
One of the main areas in which genetic engineering technologies are applied is agriculture. The classic method of improving the quality of agricultural products is selection - a process in which, through artificial selection, individual plants or animals with certain properties are isolated and crossed for the hereditary transmission of these properties and their enhancement. This process is quite lengthy and not always really effective. Genetic engineering has the ability to endow a living organism with properties that are uncharacteristic for it, to enhance the manifestation of some existing properties or to exclude them. This happens through the introduction of new or exclusion of old genes from the DNA of the organism.
For example, a special variety of potatoes resistant to the Colorado potato beetle was bred in this way. For this, the gene of the soil Thuringian bacillus Bacillus thuringiensis, which produces a special protein that is harmful to the Colorado potato beetle, but harmless to humans, was introduced into the potato genome. The use of genetic engineering to change the properties of plants, as a rule, is done just to increase their resistance to pests, adverse environmental conditions, improve their taste and growth qualities. Intervention in the genome of animals is used to accelerate their growth and increase productivity. In agricultural products, the amount of essential amino acids and vitamins, as well as their nutritional value, is also artificially increased in this way.
The number of arguments for the use of GMF far outnumbers the possible arguments against it. Thus, GMP supporters refer in particular to high level quality control of all genetically modified products (GMP). Over a twenty-year history of using these products in different countries In the world, not a single fact of their negative impact on human health has been identified, which cannot be said about the products of traditional agriculture, in which the use of various kinds of fertilizers is inevitable, many of which are recognized as harmful to humans. Moreover, the selection that has been used in agriculture for centuries, in fact, aims at the same genetic modification of organisms, only it does this over a much longer period of time. Genetic engineering is simply capable of bringing the necessary changes to the body in a short time, and therefore the use of GMF is no more dangerous than the use of any other products bred by classical selection.
Opponents of the use of genetic engineering in agriculture appeal to the lack of research on the safety of GMFs (however, this issue is constantly being investigated), as well as to the fact that GMOs sometimes cause the extinction of certain species. For example, feral genetically modified organisms can displace populations of wild species due to greater adaptability to adverse environmental conditions.
3.2. Pharmaceutics and medicine.
The production and use of vaccines against viral diseases made it possible for physicians to completely eradicate epidemics of plague and smallpox, from which millions of people used to die. The method of genetic engineering, unlike other methods, makes it possible to obtain an absolutely harmless (not containing an infectious principle) vaccine. Work is also underway to produce vaccines against influenza, hepatitis and other human viral diseases.
The services of genetic engineering are especially successfully used by pharmacists, for whom this method provides relatively cheap, but vital hormones, such as insulin, interferon, growth hormones, and others of a protein nature. By order of pharmacists, genetic engineers have launched the production of the human hormone insulin (instead of the previously used animal insulin), which plays an important role in the fight against diabetes. By the method of genetic engineering, a fairly cheap and pure human interferon is also obtained - a protein with a universal antiviral effect, an antigen of the hepatitis B virus.
Currently, Escherichia coli (E. coli) has become a supplier of such important hormones as insulin and somatotropin. Previously, insulin was obtained from animal pancreatic cells, so 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 obtain. a wide range 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 they are connected by disulfide bonds, native double-chain insulin is formed. It has been shown to be free of E. coli proteins, endotoxins and other impurities, 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 obtained 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 growth hormone with the help of 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 insulin 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.
Practical use. Now they already know how to synthesize genes, and with the help of such synthesized genes introduced into bacteria, a number of substances are obtained, in particular hormones and interferon. Their production constituted an important branch of biotechnology. Interferon, a protein synthesized by the body in response to a viral infection, is now being studied as a possible treatment for cancer and AIDS. It would take thousands of liters of human blood to produce the amount of interferon that only one liter of bacterial culture produces. It is clear that the gain from the mass production of this substance is very large. Insulin, obtained from microbiological synthesis, which is necessary for the treatment of diabetes, also plays a very important role. A number of vaccines have also been genetically engineered and are being tested to test their effectiveness against the human immunodeficiency virus (HIV), which causes AIDS. With the help of recombinant DNA, human growth hormone is also obtained in sufficient quantities, the only treatment for a rare childhood disease - pituitary dwarfism. Another promising area in medicine associated with recombinant DNA is the so-called. gene therapy. In these works, which have not yet left the experimental stage, a genetically engineered copy of a gene encoding a powerful antitumor enzyme is introduced into the body to fight a tumor. Gene therapy has also begun to be used to combat hereditary disorders in the immune system. Agriculture has succeeded in genetically modifying dozens of food and fodder crops. In animal husbandry, the use of biotechnologically produced growth hormone has increased milk yields; using a genetically modified virus created a vaccine against herpes in pigs.
4. Cloning.
The basis for the emergence of one of the most promising biomedical trends in cell replacement therapy, therapeutic cloning, was two major discoveries of the late 20th century. This is, firstly, the creation of a cloned sheep Dolly, and secondly, the receipt of embryonic stem cells (ESCs ).
Cloning is the reproduction of a living being of its non-sex (somatic) cells. Cloning of organs and her is the most important task in the field of transplantology, traumatology and other areas of medicine and biology. When transplanting cloned organs, there are no rejection reactions and there are no possible adverse consequences (for example, cancer that develops against the background of immunodeficiency). Cloned organs are a salvation for people who have been in car accidents or other disasters, as well as those who need radical help due to any disease. Cloning can give childless people the opportunity to have their own children, help people suffering from severe genetic diseases. So, if the genes that determine any hereditary disease are contained in the chromosomes, then the nucleus of her own somatic cell is transplanted into the mother's egg, then a child will appear, devoid of dangerous genes, a copy of the mother. If these genes are contained in the mother's chromosomes, the nucleus of the father's somatic cell will be transferred to her egg cell and a healthy child will appear, a copy of the father. The further progress of mankind is largely associated with the development of biotechnology. At the same time, it must be taken into account that the uncontrolled spread of genetically engineered living organisms and products can disrupt the biological balance in nature and pose a threat to human health.
Cloning a whole organism is called reproductive. Research in this direction is still underway, but there are some successes.
The case of Dolly the sheep being cloned in the UK is widely known. This mammalian cloning experiment was carried out by a group of scientists led by Ian Wilmuth. Then the nuclei taken from the udder of the donor animal were transferred into 277 eggs. Of these, 29 embryos were formed, one of which survived. Dolly was born on July 5, 1996 and became the first mammal whose cloning was successful. The cloned animal lived for 6.5 years and died on February 14, 2003 from a progressive lung disease caused by a retrovirus. It is reported to be a common disease in sheep that are kept indoors, and Dolly was largely not taken out to graze for safety reasons.
There are some misconceptions about cloning. So cloning a person or an animal is definitely not capable of repeating consciousness. The cloned individual will not be endowed with the mind of the original organism, he will need upbringing, education, etc. Moreover, the question of the full external identity of the clone is also controversial. As a rule, a clone is not a complete copy of the original, because. when cloning, only the genotype is copied, which does not mean an unambiguous repetition of the phenotype of the organism. The phenotype is formed on the basis of certain genetic data, however, the conditions in which the clone will be grown can in some way affect its development: height, weight, physique, and some features of mental development.
In most countries of the world, any work on human reproductive cloning is prohibited. Such human cloning faces even greater ethical, religious and legal problems than therapeutic one. In principle, there is no definite public opinion on this matter, just as the world's largest religions are not able to give this phenomenon unequivocal assessment, because it goes beyond the scope of their classical teachings, and therefore requires argumentation. There are also some legal complexities, such as issues of paternity, motherhood, inheritance, marriage, and some others. The development of cloning is also unsafe for reasons of control over it, as well as a possible leakage of technology into criminal and terrorist circles. Of particular concern is the high percentage of failures in cloning, which is a danger of the emergence of human freaks.
4.1 State of research on therapeutic cloning in Russia.
Despite the boom in the great possibilities of ESCs in the treatment of various diseases, there is practically no work on therapeutic cloning in Russia so far. This is primarily due to the lack of a legislative framework for research using human oocytes and embryos. With the adoption of such laws, there is a real opportunity for Russia to develop therapeutic cloning very quickly. In our country, there are effective cellular technologies for obtaining reconstructed embryos by nuclear transplantation. In essence, the foundations of modern somatic cell nuclear transfer technologies combining microsurgery and electrofusion were developed for the first time in our country in the 80s of the last century. Efficient technologies for obtaining human ESC lines are also available.
It is possible to implement the tasks of therapeutic cloning on the basis of reproduction centers, which, in addition to their direct purpose, can become centers for obtaining ESC lines, first of all, directly for female patients of this center and any members of their families. It can be expected that with the development of therapeutic technologies, the production of own ESCs will become available to everyone. It is necessary to carry out close cooperation between reproduction centers and relevant research laboratories focused on solving fundamental problems and developing new technologies. Such technologies include the reconstruction of embryos using non-invasive optical-laser micromanipulation techniques for therapeutic cloning.
5. Problems of genetic engineering.
Genetic engineering is a completely new technology that breaks down fundamental genetic barriers not only between species, but also between people, animals and plants. By combining the genes of dissimilar and unrelated species, forever changing their genetic codes, new organisms are created that will pass on the genetic changes to their descendants. Scientists today are able to cut, paste, recombine, transform, edit, and program genetic material. Animals and even human genes are added to plants or animals, giving rise to unimaginable transgenic life forms. For the first time in history, human beings have become the architects of life. Bioengineers will be able to create tens of thousands of new organisms over the next few years. The prospects are daunting. Genetic engineering raises unprecedented ethical and social questions, as well as endangering the well-being of the environment, human and animal health, and the future of agriculture. The following describes just a few of the problems associated with genetic engineering:
Genetically modified organisms that escape or are released from the laboratory can cause environmental destruction. Genetically engineered "biological pollutants" have the potential to be more destructive than even chemical pollutants. Because they are living, genetically modified products are inherently more unpredictable than chemical ones—they can reproduce, migrate, and mutate. Once these genetically modified organisms are released on Wednesday, it will be almost impossible to get them back into the lab. Many scientists warn that the release of such organisms into the environment can lead to irreversible destructive consequences for the environment.
Genetic changes are likely to lead to unintended results and dangerous surprises. Biotechnology is an inexact science and scientists can never guarantee 100 percent success. In practice, there were serious cases. Researchers conducting experiments at the University of Michigan recently found that genetically modified plants that are resistant to viruses can cause viruses to mutate into new, more dangerous forms or forms that can attack other plant species. Other frightening scenarios: Alien genes from genetically modified plants can be transferred along with pollen, insects, wind or rain to other crops, as well as wild and weed plants. Trouble can happen if the properties of genetically modified crops, such as resistance to viruses or insects, get weeds, for example. Genetically modified plants are capable of producing toxins and other substances that can harm birds and other animals. Genetic engineering of plants and animals will almost certainly endanger species and reduce biodiversity. Due to their "superior" genes, some of the plant and animal GIs will inevitably spiral out of control, conquering wild species. This has already happened when exotic species are imported into the country, for example, in North America there have been problems with Dutch Elm Disease and Pueraria Curlis. What will happen to wild species, for example, when scientists release on Wednesday a carp, salmon or trout twice as big and eating twice as much food as their wild relatives? Another danger lies in the creation of new types of crops and domestic animals. Once scientists create what will be called the “perfect tomato” or “perfect chicken,” they will be reproduced in large numbers; "less desirable" species will be left at the curb. The "ideal" animals and plants would then be cloned (reproduced as exact genetic copies), further reducing the base of available genes on the planet.
Genetic modification of crops and animals can provoke the development of toxic and allergic reactions in people. A person who is allergic to nuts or shellfish, for example, will have no way of knowing if a tomato or other food has been altered with the proteins of allergenic foods, and therefore consuming these GI foods can be fatal. In addition, genetic engineers can take a protein from a bacterium found in the soil, the ocean - anywhere - and add it to human food. Such substances have never been added to food before, so there is no information about their toxicity and allergenicity.
There are cases when genetically modified products harmed people. In 1989 and 1990, genetically engineered L-tryptophan, a common food supplement, killed more than 30 Americans and permanently disabled more than 5,000 people with the potentially fatal and painful blood disorder, eosinophilia-myalgia syndrome, before being banned. Showa Denko K.K., Japan's third largest chemical company, used a genetically engineered bacterium to create this over-the-counter supplement. It is believed that the bacterium was somehow infected through the process of DNA recombination. There is no indication on the products that it has been genetically modified. The patenting of GI products and the widespread production of biotech products will destroy farming as it has been known since ancient times. If this trend is not stopped, the patenting of transgenic plants and animals in the meat and dairy industry will soon lead to the development of rent-based farming, where farmers will rent plants and animals from biotech conglomerates and pay for seeds and offspring. Ultimately, within the next few decades, agriculture will be wiped out and taken over by industrial biosynthesis factories controlled by chemical and biotech companies. Never again will people enjoy natural fresh food. Hundreds of millions of farmers and other workers around the world will lose their earnings. The sustainable agricultural system will be destroyed.
Genetic modification and patenting of animals will reduce the status of living beings to manufactured products and lead to more suffering. In January 1994, it was announced that a complete map of the genome of cows and pigs had been elucidated, which preceded further development experiments on animals. In addition to the inherent cruelty of such experiments (wrong specimens were born with painful defects, lame, blind, etc.), these "production" creations did not have greater value for their "creators" than mechanical inventions. Animals genetically engineered for use in laboratories, such as the infamous "Harvard mouse", which had a human cancer-causing gene that was passed on to all subsequent generations, were made to suffer. A purely reductionist science, biotechnology reduces the significance of life to bits of information (the genetic code) that can be taken apart and put back together as one pleases. Stripped of their uniqueness and intimacy, animals that are mere objects to their "inventors" will be treated as such. Patents for more than 200 genetically modified "bizarre" animals are currently pending.
Genetically engineered organisms have never been adequately or properly tested for safety. To date, there is no appropriate governmental organization established to deal with this radical new class of creatures, potentially posing huge threats to health and environment. FDA policy and medicines The US regarding genetically modified foods illustrates the problem. In May 1992, the country developed a new policy on biotech products: genetically modified products will not be considered separately from natural ones; they will not be tested for safety; they will not contain a label indicating that they have been genetically modified; the US government will not track GI foods. As a result, neither the government nor consumers will know which whole or processed foods have been genetically modified. Vegetarians and people who exclude certain foods from their diet due to religious beliefs will face the prospect of involuntary consumption of vegetables and fruits containing animal and even human genetic material. And the health effects will only be found out through trial and error – by consumers.
By patenting the genes and living organisms they discover, a small corporate elite will soon control the entire genetic heritage of the planet. Scientists who “discover” genes and how to manipulate them can obtain patents—and thus ownership—not only for genetic modification technologies, but for the genes themselves. Chemical, pharmaceutical and biotechnology companies such as DuPont, Upjohn, Bayer, Dow, Monsanto, Cib-Geigy and Rhone-Poulenc are urgently trying to identify and patent plant and animal genes and people to complete the takeover of agriculture, animal husbandry and food production. These are the same companies that once promised a carefree life with pesticides and plastic. Can we trust their plans for the future?
The study of the human genome can lead to the declassification of personal information and new levels of discrimination. Some people are already denied health insurance based on bad genes. Won't employers demand genetic scanning, and won't they refuse their employees a job based on the results? Will the government get access to our personal genetic profiles? It's easy to imagine new level discrimination directed against those whose genetic profiles indicate that they are, for example, less intelligent or predisposed to certain diseases.
Genetic engineering has already been used to "enhance" human race, is a practice called eugenics. Gene scanning already allows us to find out if a fetus carries the genes for certain hereditary diseases. Will we start discarding fetuses in the near future on the basis of non-life-threatening defects such as myopia, predisposition to homosexuality, or for purely cosmetic reasons? Researchers at the University of Pennsylvania have applied for a patent on the GI of animal sperm cells so that properties passed from one generation to the next can be changed; this suggests that the same is possible for humans. The transition from animal eugenics to human eugenics is just one small step. Everyone wants the best for their children, but where do we stop? Inadvertently, we may soon repeat the Nazi efforts to create a "perfect" race.
The US military is building an arsenal of genetically modified biological weapons. Although the creation of offensive biological weapons has been declared illegal under international treaties, the US continues to develop such weapons for defensive purposes. However, genetically modified biological agents are identical whether they are used defensively or offensively. Areas of research for such weapons include: bacteria resistant to all antibiotics; more sustainable and dangerous bacteria and viruses that live longer and kill faster, as well as new organisms that can nullify the effect of a vaccine or reduce the natural resistance of people and plants. The possibility of developing pathogenic microorganisms that can disrupt hormonal balance a person is enough to cause death, and the transformation of harmless bacteria (such as those found in the human gut) into killers. Some experts believe that GI pathogens are also being developed that target certain racial groups.
Not all scientists are optimistic about genetic engineering. Skeptics include Irwin Chargoff, an eminent biochemist who is often called the father of molecular biology. He warns that not all innovation leads to "progress". Chargoff once called genetic engineering a "molecular Auschwitz" and warned that the technology of genetic engineering poses a greater threat to the world than the advent of nuclear technology. “I feel that science has crossed a barrier that should remain intact,” he wrote in his autobiography. Noting the "terrifying irreversibility" of the planned genetic engineering experiments, Chargoff warned that "... you cannot cancel a new form of life... it will outlive you, and your children, and your children's children. An irreversible attack on the biosphere is something so unheard of, so unimaginable to previous generations, that I can only wish that it was not my fault."
5. Conclusion
Public opinion. Despite the clear benefits of genetic research and experimentation, the very concept of "genetic engineering" has given rise to various suspicions and fears, has become a matter of concern and even political controversy. Many fear, for example, that some virus that causes cancer in humans will be introduced into a bacterium that normally lives in the body or on the skin of a person, and then this bacterium will cause cancer. It is also possible that a plasmid carrying a drug resistance gene will be introduced into pneumococcus, causing the pneumococcus to become resistant to antibiotics and the pneumonia to be untreatable. Such dangers certainly exist. genetic research are conducted by serious and responsible scientists, and methods to minimize the possibility of accidental spread of potentially dangerous microbes are constantly being improved. Assessing the possible dangers that these studies conceal, they should be compared with the real tragedies caused by malnutrition and diseases that kill and maim people.
Genetic engineering is one of the most actively developing and promising technologies of our time, which in the future will be able to solve many problems in medicine and not only. My personal opinion on most of the contentious issues of genetic engineering is leaning towards allowing research and application of these technologies.
In my opinion, the genetic modification of organisms, with reasonable control over this process, can solve some of the serious problems of our time. In particular, the use of genetic modification in medicine for the purpose of treating various diseases seems to me a positive phenomenon that does not cause any complaints about this stage development of science.
As for the use of genetic modification in agriculture and the distribution of genetically modified products, in my opinion, their hypothetical danger to human health is not actually confirmed. It seems to me that if the standard safety studies of these products indicate that their use is possible, then they do not need any additional research. GMOs in this case should be considered as a new type of plant or product, and provided that it meets all standard food safety standards, its use should be explicitly allowed. I also share the point of view that, due to the special control to them, the improvement of their properties at the gene level and the absence of the need to use various fertilizers harmful to humans during cultivation, they can be even safer than conventional agricultural products.
Cloning issues present serious ethical issues when it comes to human cloning. At this stage, the arguments about the need for reproductive cloning of people, in my opinion, are not convincing enough, and therefore the ban on reproductive cloning seems to me justified. However, this does not mean that all research in this area should be stopped, because in the event that science can give a high probability of the survival of clones, and the public can solve other controversial issues, reproductive cloning may well be allowed.
The issue of therapeutic cloning is also quite complicated, because in order to obtain stem cells, it is necessary to stop the development of an embryo, which, in principle, can develop into a child. It seems to me that this ethical problem is in some way close to the problem of abortion. However, all things considered, I am inclined to favor allowing therapeutic cloning because this can save a person's life at the cost of a possible life interrupted at the stage of inception.
As for the very study and research of cloning issues, in particular the issues of reproductive cloning of animals, in my opinion, it should be allowed, since it is unreasonable to prohibit it in the context of using animals in any other types of laboratory research.
Bibliography.
1. A. I. Bochkarev, T. S. Bochkareva, S. V. Saxonov, Concepts of modern natural science: a textbook for university students; ed. prof. A. I. Bochkareva. - Tolyatti: TGUS, 2008. - 386 p.
2. G96Guseykhanov M.K., Radjabov O.R. Concepts of modern natural science: Textbook. - 6th ed., revised. and additional - M.: Publishing and Trade Corporation "Dashkov and Co", 2007. - 540 p.
This handbook contains all the theoretical material on the biology course required to pass the exam. It includes all elements of the content, checked by control and measuring materials, and helps to generalize and systematize knowledge and skills for the course of the secondary (complete) school.
The theoretical material is presented in a concise, accessible form. Each section is accompanied by examples of test tasks that allow you to test your knowledge and the degree of preparedness for the certification exam. Practical tasks correspond to the USE format. At the end of the manual, answers to tests are given that will help schoolchildren and applicants to test themselves and fill in the gaps.
The manual is addressed to schoolchildren, applicants and teachers.
Cell engineering is a direction in science and breeding practice that studies methods of hybridization of somatic cells belonging to different types, the possibility of cloning tissues or whole organisms from individual cells.
One of the common methods of plant breeding is the haploid method - obtaining full-fledged haploid plants from sperm or eggs.
Hybrid cells have been obtained that combine the properties of blood lymphocytes and tumor, actively proliferating cells. This allows you to quickly and in the right quantities to obtain antibodies.
tissue culture - used to obtain in the laboratory plant or animal tissues, and sometimes whole organisms. In crop production, it is used to accelerate the production of pure diploid lines after treatment of the original forms with colchicine.
Genetic Engineering- artificial, purposeful change in the genotype of microorganisms in order to obtain cultures with predetermined properties.
Main Method- isolation of the necessary genes, their cloning and introduction into a new genetic environment. The method includes the following work steps:
- isolation of the gene, its combination with the DNA molecule of the cell, which can reproduce the donor gene in another cell (inclusion in the plasmid);
– introduction of a plasmid into the genome of a bacterial cell – a recipient;
– selection of necessary bacterial cells for practical use;
– research in the field of genetic engineering extends not only to microorganisms, but also to humans. They are especially relevant in the treatment of diseases associated with disorders in the immune system, in the blood coagulation system, in oncology.
Cloning . From a biological point of view, cloning is the vegetative reproduction of plants and animals, the offspring of which carries hereditary information identical to the parent. In nature, plants, fungi, protozoa are cloned, i.e. organisms that reproduce vegetatively. In recent decades, this term has been used when the nuclei of one organism are transplanted into the egg of another. An example of such cloning was the famous sheep Dolly, obtained in England in 1997.
Biotechnology- the process of using living organisms and biological processes in the production of medicines, fertilizers, biological plant protection products; for biological treatment Wastewater, for the biological extraction of valuable metals from sea water, etc.
The inclusion in the genome of Escherichia coli of the gene responsible for the formation of insulin in humans made it possible to establish the industrial production of this hormone.
Agriculture has succeeded in genetically modifying dozens of food and fodder crops. In animal husbandry, the use of biotechnologically produced growth hormone has increased milk yields;
using a genetically modified virus to create a vaccine against herpes in pigs. With the help of newly synthesized genes introduced into bacteria, a number of the most important biologically active substances are obtained, in particular hormones and interferon. Their production constituted an important branch of biotechnology.
With the development of genetic and cell engineering, there is more and more concern in society about the possible manipulation of genetic material. Some concerns are theoretically justified. For example, it is impossible to exclude the transplantation of genes that increase resistance to antibiotics of some bacteria, the creation of new forms of food products, but these works are controlled by governments and society. In any case, the danger from disease, malnutrition and other shocks is much higher than from genetic research.
Prospects for Genetic Engineering and Biotechnology:
- the creation of organisms useful to humans;
– obtaining new drugs;
– correction and correction of genetic pathologies.
EXAMPLES OF TASKS
Part A
A1. The production of drugs, hormones and other biological substances is engaged in such a direction as
1) genetic engineering
2) biotech production
3) agricultural industry
4) agronomy
A2. When would tissue culture be the most useful method?
1) upon receipt of a hybrid of apple and pear
2) when breeding pure lines of smooth-seed peas
3) if necessary, transplant the skin to a person with a burn
4) upon receipt of polyploid forms of cabbage and radish
A3. In order to artificially obtain human insulin by genetic engineering methods on an industrial scale, it is necessary
1) introduce a gene responsible for the synthesis of insulin into bacteria that will begin to synthesize human insulin
2) inject bacterial insulin into the human body
3) artificially synthesize insulin in a biochemical laboratory
4) grow a cell culture of the human pancreas responsible for the synthesis of insulin.
Part WITH
C1. Why are many in society afraid of transgenic products?
- 3.2. Reproduction of organisms, its significance. Methods of reproduction, similarities and differences between sexual and asexual reproduction. The use of sexual and asexual reproduction in human practice. The role of meiosis and fertilization in ensuring the constancy of the number of chromosomes in generations. Application of artificial insemination in plants and animals
» book title
Traditional selection of microorganisms (mainly bacteria and fungi) is based on experimental mutagenesis and selection of the most productive strains. But even here there are some peculiarities. The genome of bacteria is haploid, any mutations appear already in the first generation. Although the probability of a natural occurrence of a mutation in microorganisms is the same as in all other organisms (1 mutation per 1 million individuals for each gene), a very high reproduction rate makes it possible to find a useful mutation for the gene of interest to the researcher.
As a result of artificial mutagenesis and selection, the productivity of penicillium fungus strains was increased by more than 1000 times. Products of the microbiological industry are used in baking, brewing, winemaking, and the preparation of many dairy products. With the help of the microbiological industry, antibiotics, amino acids, proteins, hormones, various enzymes, vitamins and much more are obtained.
Microorganisms are used for biological wastewater treatment, improving soil quality. At present, methods have been developed for obtaining manganese, copper, and chromium in the development of dumps of old mines with the help of bacteria, where conventional mining methods are economically unprofitable.
Biotechnology- the use of living organisms and their biological processes in the production of substances necessary for man. The objects of biotechnology are bacteria, fungi, cells of plant and animal tissues. They are grown on nutrient media in special bioreactors.
The latest breeding methods microorganisms, plants and animals are cellular, chromosomal and genetic engineering.
Genetic Engineering
Genetic Engineering- a set of techniques that allow you to isolate the desired gene from the genome of one organism and introduce it into the genome of another organism. Plants and animals in which "alien" genes are introduced into the genome are called transgenic, bacteria and fungi transformed. The traditional object of genetic engineering is Escherichia coli, a bacterium that lives in the human intestine. It is with its help that growth hormone is obtained - somatotropin, the hormone insulin, which was previously obtained from the pancreas of cows and pigs, interferon protein, which helps to cope with a viral infection.
The process of creating transformed bacteria includes the following steps.
- Restriction- “cutting out” the necessary genes. It is carried out with the help of special "genetic scissors", enzymes - restrictase.
- Create a vector- a special genetic construct, in which the intended gene will be introduced into the genome of another cell. The basis for creating a vector are plasmids. The gene is sewn into the plasmid using another group of enzymes - ligases. The vector must contain everything necessary to control the operation of this gene - a promoter, a terminator, an operator gene, and a regulator gene, as well as marker genes that give the recipient cell new properties that make it possible to distinguish this cell from the original cells.
- Transformation- introduction of the vector into the bacterium.
- Screening- selection of those bacteria in which the introduced genes work successfully.
- Cloning transformed bacteria.
1 - cell with the original plasmid; 2 - isolated plasmid; 3 - vector creation; 4 — recombinant plasmid (vector); 5 — cell with recombinant plasmid.
Eukaryotic genes, unlike prokaryotic ones, have a mosaic structure (exons, introns). IN bacterial cells there is no processing, and translation in time and space is not separated from transcription. In this regard, it is more efficient to use artificially synthesized genes for transplantation. The template for such synthesis is mRNA. With the help of the enzyme reverse transcriptase, a DNA chain is first synthesized on this mRNA. Then a second strand is completed on it with the help of DNA polymerase.
Chromosomal engineering
Chromosomal engineering- a set of techniques that allow manipulations with chromosomes. One group of methods is based on the introduction into the genotype of a plant organism of a pair of foreign homologous chromosomes that control the development of the desired traits ( complemented lines), or substitution of one pair of homologous chromosomes for another ( replaced lines). In the thus obtained substituted and supplemented lines, traits are collected that bring plants closer to the "ideal variety".
haploid method based on the cultivation of haploid plants with subsequent doubling of chromosomes. For example, haploid plants containing 10 chromosomes are grown from corn pollen grains ( n= 10), then the chromosomes are doubled and get diploid ( n= 20), fully homozygous plants in just 2-3 years instead of 6-8 years of inbreeding.
This can also include method for obtaining polyploid plants(see Lecture 23 Plant Breeding).
Cell engineering
Cell engineering— construction of a new type of cells based on their cultivation, hybridization and reconstruction.
Cells of plants and animals, placed in nutrient media containing all the substances necessary for life, are able to divide, forming cell cultures. Plant cells also have the property totipotency, that is, under certain conditions, they are able to form a full-fledged plant. Therefore, it is possible to propagate plants in test tubes by placing the cells in certain nutrient media. This is especially true for rare or valuable plants.
With the help of cell cultures, it is possible to obtain valuable biologically active substances (ginseng cell culture). Obtaining and studying hybrid cells allows solving many problems of theoretical biology (mechanisms of cell differentiation, cell reproduction, etc.). Cells obtained as a result of the fusion of protoplasts of somatic cells belonging to different species (potato and tomato, apple and cherry, etc.) are the basis for creating new forms of plants. In biotechnology, monoclonal antibodies are used hybridomas- a hybrid of lymphocytes with cancer cells. Hybridomas produce antibodies, like lymphocytes, and have the ability to multiply in culture indefinitely, like cancer cells.
The method of transplanting the nuclei of somatic cells into eggs allows you to get a genetic copy of the animal, that is, it makes it possible cloning animals. At present, cloned frogs have been obtained, and the first results of cloning mammals have been obtained.
The method of fusion of embryos in the early stages makes it possible to create chimeric animals. In this way, chimeric mice were obtained (fusion of embryos of white and black mice), a chimeric animal sheep-goat.
For the treatment of many diseases, various biologically active substances are needed. When they are isolated from human tissues, there is a danger of contamination of the obtained material with various viruses (hepatitis B, human immunodeficiency, etc.). In addition, these substances are produced in small quantities and are expensive. Biologically active substances of animal origin are ineffective due to incompatibility with the immune system of a sick person. Only the development of a new branch of genetic engineering helped to ensure the production of pure biologically active substances in large quantities at a lower cost.
Genetic Engineering- this is the creation of hybrid, recombinant DNA molecules, and therefore, organisms with new features. To do this, it is necessary to isolate a gene from any organism or artificially synthesize it, clone (multiply) and transfer it to another organism.
The tools of genetic engineering are enzymes: restriction enzymes (cutting the DNA molecule) and ligases (crosslinking it). Viruses are used as carrier vectors.
With the help of genetic engineering, strains of Escherichia coli were created, in which the genes for human insulin (necessary for the treatment of diabetes mellitus), interferon ( antiviral drug), somatotropin (growth hormone).
Genetically engineered yeast cells that produce human insulin. The biosynthetic method for the production of human insulin using yeast cells is widely used in pharmaceutical production (in Denmark, Yugoslavia, the USA, Germany and other countries).
At present, scientists from different countries are working on obtaining, using genetic engineering, a number of other necessary biologically active substances, a vaccine against hepatitis B, a profibrinolysin activator (an anticoagulant drug), interleukin-2 (an immunomodulator), etc.
Foreign genes are introduced into animal cells in the form of individual DNA molecules or as part of virus vectors capable of introducing foreign DNA into the cell genome. Usually two methods are used:
1) DNA is added to the cell incubation medium;
2) DNA microinjections are made directly into the nucleus (which is more efficient).
The primary tasks of genetic engineering in humans are the creation of human gene banks for their study and the search for ways of gene therapy, that is, the replacement of mutant genes with normal alleles.
Cell engineering is a method of constructing a new type of cell based on their cultivation, hybridization or reconstruction. In hybridization, whole cells (sometimes of distant species) are artificially combined to form a hybrid cell. Cellular reconstruction is the creation of a viable cell from separate fragments of different cells (nucleus, cytoplasm, chromosomes, etc.).
The study of hybrid cells makes it possible to solve many problems in biology and medicine. So, for example, biotechnology uses hybridomas. hybridoma is a cell hybrid obtained by the fusion of a normal lymphocyte and a tumor cell. It has the ability to synthesize monoclonal (homogeneous) antibodies of the desired specificity (property of a lymphocyte) and to unlimited growth in an artificial environment (property of a tumor cell).
Biotechnology- is the production of products and materials necessary for humans, with the help of biological objects.
The term "biotechnology" became widespread in the mid-70s of the XX century, although certain branches of biotechnology have been known for a long time and are based on the use of various microorganisms: baking, winemaking, brewing, cheese making. Advances in genetics have created great additional opportunities for the development of biotechnology.
In the middle of the XX century. and in the second half, using induced mutagenesis, antibiotics were obtained (penicillin, streptomycin, erythromycin, etc.) - with the help of microbes; amylase enzyme - with the help of hay bacillus, amino acids - with the help of Escherichia coli; lactic acid - with the help of lactic acid bacteria; lemon acid- with the help of Aspergillus mold; B vitamins - with the help of yeast.
Loading...