At the above three levels, the proliferation and separation of a single clonal population is called cloning. At this point, the word clone can also be understood as a verb. Cloning is the core part of recombinant DNA technology. In fact, cloning technology has been used to propagate viruses, other microorganisms and pure plants through nutrition, thus ensuring the accurate continuity of the genomes of these organisms. Now, the word cloning also includes the separation and preservation of an independent genetic factor. The cloning of cell organisms only needs nutrient medium, while the cloning of genes needs some vector replicons, specific host cells and nutrient medium. Cloning technology of various organisms plays an important role in bioengineering.
"clone" is a transliteration of the English word "clone", which has three different meanings in the field of biology.
1. At the molecular level, cloning generally refers to DNA cloning (also called molecular cloning). It refers to inserting specific DNA fragments into vectors (such as plasmids and viruses) by recombinant DNA technology, and then replicating in host cells to obtain a large number of identical DNA fragments "groups".
2. At the cellular level, cloning is essentially a cell group formed by the division of a single ancestor cell. These cells all have the same gene. For example, a cell group with the same genetic background formed by dividing a cell in vitro culture medium for several generations is a cell clone. For another example, in vertebrates, when foreign substances (such as bacteria or viruses) invade, specific recognition antibodies will be produced through immune response. All plasma cells that produce specific antibodies are formed by B cell division, and such plasma cell groups are also cell clones. Cell cloning is a low-level reproductive mode-asexual reproduction, that is, offspring and parents have the same heredity and do not need sexual union. The lower the level of biological evolution, the more likely it is to adopt this mode of reproduction.
3. At the individual level, cloning refers to a group of two or more individuals with the same genotype. For example, two identical twins are a clone! Because they come from the same egg cell, their genetic background is exactly the same. According to this definition, "Dolly" cannot be said to be a clone! Because "Dolly" is just a lonely one. Only by transplanting two or more identical nuclei into two or more identical enucleated eggs and obtaining two or more Dollies with the same genetic background can British embryologists use the word cloning. Therefore, in the sensational paper published in Nature from 65438 to February 1997, the author did not describe Dolly as a clone.
In addition, cloning can also be used as a verb, which refers to the above-mentioned process of obtaining DNA, cells or individual groups.
Second, cloning technology.
1.DNA clone
At present, there are various methods of DNA cloning, and the basic flow is shown in the following figure (not to scale).
Thus, the DNA thus obtained can be applied to many aspects of biological research, including the analysis and processing of the base sequence of specific DNA, and the mass production of valuable protein in biotechnology industry.
2. Cloning of biological individuals
(1) Cloning of Plant Individuals
In 1950s, botanists used carrots as model materials to study whether genetic material was lost in differentiated plant cells. To their surprise, they found that from a highly differentiated carrot cell,
Can develop into a complete plant! Therefore, they think that plant cells are omnipotent. The carrot population developed from more than two somatic cells in a carrot has the same genetic background, so it is a clone. The cloning process of this plant is a complete asexual reproduction process!
(2) Cloning of individual animals
(1) The Birth of "Dolly"
1On February 27th, 997, Ian Wilmott of Roslin Institute in Edinburgh, England, announced to the world that Dolly, the world's first cloned sheep, was born, which immediately caused a sensation all over the world.
Dolly is related to three ewes. One is a Dorset ewe in Finland who is pregnant for three months, and the other is a black-faced ewe in Scotland. Dorset ewes in Finland provided a complete set of genetic information, that is, provided the nucleus (called donor); Scottish black-faced ewes provide eggs without nuclei; Another Scottish black-faced ewe provides the development environment of sheep embryos-uterus, which is Dolly's "biological" mother. The whole cloning process is briefly described as follows:
Breast cells were taken from the mammary glands of Dorset ewes in Finland and put into low concentration nutrient medium, and the cells gradually stopped dividing, which was called donor cells. Inject gonadotropin into Scottish black-faced ewe to induce ovulation, take out unfertilized egg cells, and immediately enucleate them, leaving a seedless egg cell called recipient cell; Using the method of electric pulse, donor cells and recipient cells are fused, and finally a fused cell is formed. Because electric pulses can also produce a series of reactions similar to the natural fertilization process, fused cells can also divide and differentiate like fertilized eggs, thus forming embryonic cells. Embryonic cells were transplanted into the uterus of another Scottish black-faced ewe, and the embryonic cells further differentiated and developed, eventually forming a little sheep. Dolly sheep born are exactly the same as Dorset ewes in appearance.
A year later, another group of scientists reported that more than 20 fully developed mice were obtained by transplanting the nucleus of mouse cumulus cells (highly differentiated cells on the periphery of oocytes) into oocytes without nuclei. If Dolly is not enough to be called a cloned sheep, because there is only one, these mice.
It is a veritable cloned mouse.
② The basic process of cloning mice by nuclear transfer.
In this experiment, cumulus cells were obtained through the following process: by injecting chorionic gonadotropin repeatedly, female rats were induced to enter a state of high egg production. Then the complex of cumulus cells and oocytes was collected from the fallopian tubes of female rats. Hyaluronic acid treatment dispersed cumulus cells. Cumulus cells with a diameter of 10- 12 micron were selected as nuclear donors (previous experiments showed that if cumulus nuclei with smaller or larger diameters were used, oocytes after nuclear transfer rarely developed to 8-cell stage). The selected cumulus cells were stored in a certain solution environment and nuclear transplantation was carried out within 3 hours (unlike this, when Dolly was obtained, the breast cells used as nuclear donors were propagated in the culture solution for 3-6 times).
Oocytes were collected from different female mice (usually in metaphase II of meiosis) by a method similar to that described above. Carefully take out the nucleus of the oocyte with a thin tube with a diameter of about 7 microns under the microscope, and try not to take out the cytoplasm. Also be careful to remove the nucleus of cumulus cells and try to remove the cytoplasm (remove a small amount of cytoplasm by reciprocating the nucleus in the glass tube several times). Within 5 minutes after enucleation, it was directly injected into enucleated oocytes. The nuclear transferred oocytes were placed in a special solution for 1-6 hours, and then bivalent strontium ions (Sr2+) and cytochalasin b were added. The former activates oocytes, while the latter inhibits the formation of polar bodies and the elimination of chromosomes. Then, the treated oocytes were taken out and put into a special solution without strontium and cytochalasin B to divide the cells into embryos.
Embryos of different stages (from 2- cell stage to blastocyst stage) were implanted into the fallopian tubes or uterus of pseudopregnant female mice that mated with ligated male mice a few days ago for development. After about 19 days, the fully developed fetal rats were taken out by surgery.
At present, animals cloned by embryo nuclear transfer include mice, rabbits, goats, sheep, pigs, cows and monkeys. In China, there are cloned animals except monkeys, and goats can also be cloned through continuous nuclear transfer. This technology goes further than embryo segmentation technology, and more animals will be cloned. Because the more times the embryo divides, the fewer cells there are in each part, and the worse the individual ability is. There is only one kind of animal cloned by somatic cell nuclear transfer, that is Dolly sheep.
Third, the gospel of cloning technology.
1. Cloning Technology and Genetic Breeding
In agriculture, people have cultivated a large number of high-quality and high-yield varieties with drought resistance, lodging resistance and pest resistance by using "cloning" technology, which has greatly increased grain output. In this regard, China has entered the forefront of the world's most advanced.
2. Cloning technology and protection of endangered species
Cloning technology is a gospel for protecting species, especially rare and endangered species, and has great application prospects. From a biological point of view, this is also one of the most valuable places of cloning technology.
3. Cloning technology and medicine
At present, doctors can transplant almost all human organs and tissues. But as far as science and technology are concerned, rejection in organ transplantation is still the most troublesome. The reason of rejection is poor compatibility due to tissue mismatch. If the organs of "clones" are provided to "hominids" for organ transplantation, there is no need to worry about rejection at all, because the genes and tissues of the two are matched. The question is, is it humane to use "clones" as organ donors? Is it legal? Is it economical?
Cloning technology can also be used to multiply valuable genes. For example, in medicine, people produce insulin to treat diabetes, growth hormone to make dwarfism patients grow taller again, fibrinolytic enzyme to resist various virus infections and so on through "cloning" technology.
Interviewee: Anonymous 3- 16 19:06.
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Cloning is a transliteration of English clone, which is simply an artificially induced asexual reproduction method. But cloning is different from asexual reproduction. Asexual reproduction means that there is no combination of male and female germ cells, and only one kind of organism produces offspring. The common reproduction methods are spore reproduction, budding reproduction and fission reproduction. By layering, cutting or grafting the roots, stems and leaves of plants to produce new individuals, it is also called asexual reproduction. Sheep, monkeys, cows and other animals cannot reproduce asexually without manual operation. Scientists call artificial gene manipulation of animal and plant reproduction process cloning, and this biotechnology is called cloning technology.
The idea of cloning technology was first put forward by German embryologist in 1938. 1952, scientists first carried out cloning experiments with frogs, and then people continued to study cloning technology with various animals. Because of the little progress in this technology, the research work once entered a trough in the early 1980 s, and later some people cloned it successfully with mammalian embryonic cells. 1On July 5th, 1996, British scientist Dr Ian Wilmut cloned a live sheep from adult sheep somatic cells, which brought a major breakthrough in the research of cloning technology. It broke through the technical difficulty that only embryonic cells could be used for animal cloning in the past, and achieved the goal of animal cloning with somatic cells for the first time, realizing animal replication in a higher sense. The goal of studying cloning technology is to find a better way to change the genetic composition of domestic animals and cultivate animal groups that can provide consumers with better food or any chemicals they may need.
The basic process of cloning is to transplant the nucleus of a donor cell containing genetic material into an egg cell without nucleus, then fuse the two cells through micro-current stimulation, and then promote the new cells to divide and reproduce and develop into embryos. When the embryo develops to a certain extent (it takes about 6 days for Roslin Institute to clone sheep), it is implanted into the uterus of an animal to make the animal pregnant and give birth to an animal with the same gene as the donor. In this process, if the donor cells are genetically modified, the genes of the offspring of asexual animals will also change in the same way. The main difference between "Honu Lu Lu badminton technology" and Dolly sheep technology that successfully bred three generations of cloned mice is that the genetic material in the cloning process is directly injected into the egg cells by physical methods, rather than cultivated in the culture medium. In this process, chemical stimulation is used instead of electrical stimulation to control the egg cells again. 1On July 5th, 998, scientists from Ishikawa Animal Husbandry Center and Animal Husbandry Laboratory of Feng Jingen University announced that two calves cloned from adult animal somatic cells were born. The birth of these two cloned cows shows that the technology of cloning adult animals is repeatable.
1996 When Dolly was cloned by Roslin Institute in Scotland, this achievement was immediately hailed as one of the most important and controversial scientific and technological breakthroughs in this century. The benefits of this breakthrough are obvious. This technology can play an important role in rescuing rare and endangered animals, replicating excellent livestock individuals, expanding and improving animal populations, improving the genetic quality and production performance of herds, providing enough experimental animals, promoting the research of transgenic animals, overcoming genetic diseases, developing high-level new drugs, and producing internal organs for human transplantation.
While affirming the positive role of this technology, people also expressed their concerns about this technology to a greater extent. Hey? Bad looks? What is the purpose of the temple? The popularization of this asexual reproduction technology in animal husbandry is likely to destroy the ecological balance and lead to the large-scale spread of some diseases; If it is applied to human reproduction, there will be a huge ethical crisis.
After Dolly the sheep's identity was revealed, scientists in Oregon in the United States also confirmed that they had bred monkeys from cloned embryos in August 1996. There is also a legend that a doctor in Belgium accidentally cloned a boy. Although Belgian scientists denied the report of human cloning, governments all over the world attached great importance to the possible legal and ethical impact of cloning technology. The United States, Germany, France, Britain, Canada and other countries set up expert groups to study this issue, and scientists also demanded that research in this field be restricted. Nakajima Hiroshi, Director-General of the World Health Organization, and the Scientific Research Committee of the European Commission1March 997 1 1 made statements and talks respectively, expressing their opposition to human cloning experiments. At present, countries have a consensus on this technology, that is, laws should be enacted to strengthen the management of this technology, and it is strictly forbidden to use it to copy human beings. Wilmut, a British scientist who cloned Dolly sheep, also said that the technical efficiency of cloning Dolly sheep was extremely low, which had led to the birth of animals with congenital defects before he successfully cloned Dolly sheep. It is "very inhuman" to apply this technology to human beings.
China government also attaches great importance to cloning technology and related issues. The State Science and Technology Commission and the Ministry of Agriculture and other departments have held many seminars and symposiums attended by experts from all walks of life, and reached consensus on relevant issues. Experts believe that the success of animal cloning technology is a major event in scientific research, with both favorable and unfavorable possibilities. Measures must be taken to regulate and strictly control the harmful side, so that this technology can benefit mankind.
1997165438+1October 1 1 The 29th General Conference of UNESCO adopted a document entitled "Universal Declaration on Human Genome and Human Rights" in Paris, which explicitly opposed the reproduction of human beings by cloning technology. The document points out that the research of human genome should make use of the achievements of biology, genetics and medicine. However, this research must be aimed at maintaining and improving public health, and practices that violate human dignity, such as cloning human beings, are not allowed.
1998 65438+1October 12,19 European countries signed the European protocol prohibiting human cloning in Paris, France. This is the first international legal document prohibiting human cloning, which is a supplement to the European Biomedical Treaty. This agreement on the prohibition of human cloning stipulates that it is forbidden for research institutions or individuals in signatory countries to use any technology to create a person with genetic similarity to a living or dead person, otherwise they will be severely punished. Researchers and doctors who violate the agreement will be banned from conducting research and practicing medicine, and the licenses of relevant research institutes or hospitals will be revoked. If the research institutions or individuals of the signatory countries conduct such activities outside Europe, they will also be investigated for legal responsibility. France, Denmark, Lithuania, Finland, Greece, Ireland, Italy, Latvia, Luxembourg, Moldova, Norway, Portugal, Romania, Slovenia, Spain, Sweden, Macedonia, Turkey and San Marino have signed the agreement.
Development of cloning technology
Cloning is the transliteration of Clone, which means asexual reproduction, and cloning technology is asexual reproduction technology. Not long ago, it was reported that Dolly the cloned sheep was successfully cloned from somatic cells for the first time, which opened a new page in the history of bioengineering.
Cloning technology has experienced three development periods:
The first period is microbial cloning, that is, thousands of identical bacteria are copied from one bacteria and become a bacterial colony.
The second period is biotechnology cloning, such as DNA cloning.
The third period is animal cloning, that is, cloning a cell into an animal.
In nature, many plants have innate cloning instinct, such as sweet potatoes, potatoes, roses and other cutting propagation plants. However, animal cloning technology has experienced the development process from embryonic cells to somatic cells. As early as 1950s, American scientists took amphibians and fish as research objects and pioneered nuclear transplantation technology. They studied the potential of cell development and differentiation and the interaction between cytoplasm and nucleus. 1986, British scientist Wila Anderson cloned a sheep from embryonic cells for the first time through nuclear transfer, and later some people cloned animals such as cattle, sheep, rats, rabbits and monkeys. China's cloning technology is also quite successful. In the late 1980s, China cloned a rabbit. 199 1 year, Northwest Agricultural University and Development Institute of Jiangsu Agricultural University successfully cloned sheep. 1993 Institute of Developmental Biology of China Academy of Sciences and Agricultural College of Yangzhou University * * * cloned a batch of goats, 1995 South China Normal University and Guangxi Agricultural University cooperated to clone cattle. Recently, the United States successfully cloned monkeys, and Japanese scientists also claimed that they had bred more than 200 "cloned cows". All the cloned animals were successfully transplanted with embryonic cells as donor cells.
1997 In February, Roslin Institute announced the successful cloning of a lamb named Dolly. It uses mammary epithelial cells as donor cells for nuclear transfer, which opens a new page in the history of biological cloning, breaks through the traditional way of nuclear transfer using embryonic cells and makes great progress in cloning technology. The whole cloning process is as follows: scientists selected three ewes, first sucked all the genetic material from the egg cell of one ewe, and then fused the mammary gland cells of another 6-year-old ewe to form an egg cell containing new genetic material, and promoted its division and development into an embryo. When the embryo grows to a certain extent, it is implanted into the uterus of a third ewe, conceived and gives birth to Dolly, a cloned sheep. Dolly is like a 6-year-old ewe that provides breast cells. Dolly sheep is the first animal successfully cloned by somatic cells in the world. The success of Dolly's cloning theoretically shows that highly differentiated cells can restore the zygotic function of fertilized eggs after certain treatment. It shows that cytoplasm can regulate the development of heterogeneous nuclei during development. It provides an important way for the treatment of biological genetic diseases, the cultivation of excellent varieties and population expansion, and plays a certain role in species optimization, endangered animal germplasm preservation and transgenic animal population expansion. Since the successful cloning of Dolly sheep, countries all over the world have aroused strong repercussions, some of which are regarded as good news and some as disasters. The author thinks that we should support the new technology and make a breakthrough in biological cloning. The biggest advantage is that a large number of excellent livestock have been cultivated, which enriches people's material life, reduces the cost of animal husbandry, improves efficiency, and also provides some pharmaceutical raw materials to improve human immune function. Before Dolly the sheep, Roslin Institute had bred a transgenic sheep, whose milk contained raw materials for treating hemophilia, and a company bought it at a high price of 500,000 pounds. If we "replicate" this sheep in large quantities with somatic cells, we can save more patients' lives. In addition, cloning technology can be used to copy a large number of rare animals, save endangered species, adjust the ecological balance of nature and benefit mankind. Why worry? Of course, cloning technology may also bring negative effects. Some cloned animals have identical genes, and infection with a specific virus or other diseases will bring disaster. If there is no plan to clone animals, it will disturb the evolution of species and interfere with the sex ratio. This artificial control of the biological world will bring many unexpected hazards. However, this negative effect can be avoided as long as corresponding research countermeasures are taken and scientific cloning plans are made.
As for human cloning, it is a meaningless research topic. Contemporary biological history has proved that cloning technology can only reproduce creatures with the same appearance characteristics, but can't clone the original talents of the copied people. People's thinking ability is restricted by acquired. Therefore, even if someone can clone people like great leaders and scientists in history, they are just the same in appearance, but they lack the thoughts, temperament and talents of great leaders and scientists. What's the point of such cloning? As for some people who advocate human cloning to obtain human organs for medical organ transplantation, it is also not feasible. Because human cloning is first and foremost a citizen and enjoys human rights. If the clone refuses to donate organs, your inventor can't violate human rights. As for cloning headless people, it is also unrealistic, because clones must eat first and think first. It is impossible without a head. We can't cultivate a headless vegetable, can we? And the most important thing is that human cloning is not in line with the national conditions of the world. Nowadays, the population of the world is expanding rapidly, and many countries implement family planning to control population growth. In this case, how can we spend huge sums of money to do things that violate the laws of social development? As Ruettgers, the German Minister of Research and Technology, said: "Cloning human beings is not allowed and certainly will not happen." At present, cloning technology has made new progress in Britain, and they have applied this technology to human hematopoiesis. The economic background of cloning technology Dr. Ross James, the director of the British PPL company, said: "From the study of Dolly, we know that we can make a transgenic animal from a cell. We are now using this technology to produce the most important component in human blood, namely plasma. " They cooperated with Roslin Institute to study a kind of cattle and sheep with human genes. They first took plasma from animals, and then replaced human plasma. Transgenic cattle and sheep contain important components of human plasma. By raising, cloning or breeding these animals, stable, reliable and relatively cheap blood resources can be obtained. According to statistics, the annual value in Britain can reach 150. It can be said that it has benefited a lot. The prospect of cloning technology is immeasurable.