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 into one by 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 is implanted into the uterus of an animal to make the animal pregnant, and then an animal with the same gene as the cell donor can be born. 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.
Cloning technology does not require male and female mating, and does not require the combination of sperm and eggs. It only needs to extract single cells from animals, cultivate them into embryos by artificial methods, and then implant the embryos into female animals to breed new individuals. This cloned animal cultured with single cells has exactly the same characteristics as the single-cell donor and is a "replica" of the single-cell donor. Scientists in Britain and Oregon in the United States have successively cultivated "cloned sheep" and "cloned monkeys". The success of cloning technology is called "historic event and scientific innovation". Some people even think that cloning technology can be compared with the advent of the atomic bomb that year.
Cloning technology can be used to produce "cloned human" and "cloned human", which has aroused widespread concern all over the world. Is cloning sad or happy, a curse or a blessing for human beings? Materialist dialectics holds that everything in the world is a contradictory unity, which is divided into two parts. So is cloning technology. If we use cloning technology to "copy" a war madman like Hitler, what will it bring to human society? Even if it is used to "copy" ordinary people, it will bring a series of ethical problems. If cloning technology is applied to animal husbandry production, it will make fundamental changes in the cultivation and reproduction of excellent livestock breeds. If cloning technology is used in the research of gene therapy, it is very possible to overcome the persistent diseases that endanger human life and health, such as cancer and AIDS. Cloning technology, like atomic energy technology, is a double-edged sword with the hilt in human hands. Human beings should take joint action to avoid the emergence of "human cloning" and let cloning technology benefit human society.
Research status of cloning technology
First, the early research of cloning.
The word clone is a transliteration of the English word clone. As a noun, clone is usually translated as asexual clone. The genetic composition of all members in the same clone is exactly the same, unless there is mutation. Cloning of natural plants, animals and microorganisms has long existed in nature. For example, identical twins are actually clones. However, natural mammals have a very low incidence of cloning, a small number of members (generally two), lack of purpose, and are rarely used to benefit mankind. Therefore, people began to explore artificial methods to produce higher animal clones. In this way, the word cloning began to be used as a verb to refer to the behavior of artificially cultivating cloned animals.
At present, there are two main methods to produce mammalian clones: embryo segmentation and nuclear transfer. Dolly, a cloned sheep cultivated by scientists from various countries, and various cloned animals later adopted nuclear transplantation technology. The so-called nuclear transfer refers to the process of transplanting the nuclei of embryos or adult animals at different development stages into enucleated oocytes through microsurgery and cell fusion, re-forming embryos and making them mature. Different from embryo segmentation technology, nuclear transfer technology, especially continuous nuclear transfer technology, can produce an infinite number of genetically identical individuals. Because nuclear transfer is an effective method to produce cloned animals, people usually call it animal cloning technology.
The idea of cloning animals by nuclear transplantation was originally proposed by Hans? Spaemann put forward it in 1938, which he called a "strange experiment", that is, taking out the nucleus from an embryo (mature or immature) at the later stage of development and transplanting it into an egg. This idea is the basic way to clone animals now.
Starting from 1952, scientists first carried out nuclear transfer cloning experiments with frogs, and successively obtained tadpoles and adult frogs. From 65438 to 0963, the scientific research group led by Professor Tong Dizhou in China studied the nuclear transfer technology of fish embryos for the first time, and achieved success.
The preliminary results of mammalian embryo nuclear transfer research were obtained in 198 1 year-Karl? Gillman Ze and Peter? Hope uses mouse embryonic cells to cultivate normal mice. 1984，Steen？ Willaderson cloned a live-born sheep from immature embryo cells taken from sheep, and others later repeated the experiment with various animals such as cattle, pigs, goats, rabbits and macaques. 1989, Willadson obtained the second generation of cloned cattle with continuous nuclear transfer. 1994, Neil? The earliest cloned cattle came from late embryos with at least 120 cells. By 1995, nuclear transfer of major mammalian embryos was successful, including frozen and in vitro embryos; Nuclear transplantation experiments of embryonic stem cells or adult stem cells have also been tried. However, until 1995, the transfer of differentiated nuclei in adult animals has not been successful.
Second, the significance and response of Dolly's cloned sheep
The above facts show that before1February 1997, the research group of Dr. Wilmut of Roslin Institute announced the successful cultivation of somatic cloned sheep Dolly, the embryo nuclear transfer technology had made great progress. In fact, the cloned Dolly followed the whole process of embryo nuclear transfer in nuclear transfer technology, but this can not reduce Dolly's significance, because it is the first animal born by somatic cell nuclear transfer in the world, which is a major breakthrough in the field of cloning technology. This great progress means: it is proved theoretically that the differentiated animal nucleus is totipotent, just like plant cells, the genetic material in the nucleus will not change irreversibly during differentiation; Practice has proved that the technology of animal cloning with somatic cells is feasible, and countless identical cells can be used as donors for nuclear transfer, and a series of complex genetic operations can be carried out on these donor cells before they are fused with egg cells, thus providing an effective method for breeding excellent animal varieties and mass production of transgenic animals.
In theory, using the same method, people can copy "human cloning", which means it is entirely possible for dictators in science fiction to clone themselves. Therefore, the birth of "Dolly" has aroused strong repercussions in the scientific, political and even religious circles all over the world, and triggered a discussion on the moral issues derived from human cloning. Governments and people all over the world have reacted: cloning human beings is against ethics. Nevertheless, the great theoretical significance and practical value of cloning technology have prompted scientists to speed up their research, thus pushing the research and development of animal cloning technology to a climax.
Three, the important achievements of cloning research in recent three years
The birth of Dolly, a cloned sheep, set off an upsurge of cloning research all over the world. Subsequently, reports on cloned animals continued. 1 in March, 997, that is,1month after Dolly was born, scientists from the United States, China, Taiwan Province Province of China and Australia respectively announced that they had successfully cloned monkeys, pigs and cows. But all of them are cloned with embryonic cells, and the significance can't be compared with Dolly. In July of the same year, Roslin Institute and PPL announced that Polly, the world's first transgenic sheep with human genes, had been cloned from transgenic fetal fibroblasts. This achievement shows the great application value of cloning technology in cultivating transgenic animals.
In July, 1998, Wakayama, University of Hawaii reported that 27 surviving mice were cloned from mouse cumulus cells, of which 7 were only the offspring of cloned mice, which was the second batch of mammalian somatic cell nuclear transfer offspring after Dolly. In addition, Wakayama and others adopted a relatively simple and high success rate new cloning technology, which was different from Dolly's, and named it "Honolulu Technology" after the location of the university.
Since then, scientists from the United States, France, the Netherlands and South Korea have also reported the success of somatic cell cloning cattle. The research enthusiasm of Japanese scientists is particularly amazing. July 1998 to April 1999, Tokyo Agricultural University, Feng Jingen University, livestock improvement enterprise group, local livestock test sites (Ishikawa Prefecture, Oita Prefecture, Kagoshima Prefecture, etc.) and private enterprises (such as Yin Xue Dairy, the largest dairy company in Japan, etc.). It is reported that they use bovine ear and hip muscles. By the end of 1999, somatic cloned offspring of six types of cells-fetal fibroblasts, breast cells, cumulus cells, oviduct/uterine epithelial cells, muscle cells and ear skin cells-had been successfully born in the world.
In June, 2000, China Northwest A&F University cloned two "cloned sheep" from adult goat somatic cells, but one of them died of respiratory system dysplasia. According to reports, the cloning technology adopted by the research team is completely different from Dolly's, which shows that scientists in China have also mastered the cutting-edge technology of somatic cell cloning.
Nuclear transplantation experiments between different species have also achieved some gratifying results. 1998, 1 In June, scientists at the University of Wisconsin-Madison in the United States successfully cloned the embryos of five mammals: pigs, cows, sheep, rats and macaques. The results show that the unfertilized eggs of a species can combine with the mature nuclei of many animals. Although these embryos miscarried, it made a useful attempt to the possibility of heterogeneous cloning. 1999, American scientists cloned the embryo of a rare animal argali from cow eggs. Scientists in China have also cloned early embryos of giant pandas from rabbit eggs, which indicates that cloning technology may become a new way to protect and save endangered animals.
Fourth, the application prospect of cloning technology
Cloning technology has shown broad application prospects, which can be summarized in the following four aspects: (1) cultivating excellent varieties and producing experimental animals; (2) producing transgenic animals; (3) producing human embryonic stem cells for cell and tissue replacement therapy; (4) reproduction of endangered animal species, preservation and dissemination of animal species resources. The production of transgenic animals and embryonic stem cells is briefly described as follows.
The research on transgenic animals is one of the most attractive and promising topics in the field of animal bioengineering. Transgenic animals can be used as donors for medical organ transplantation, as bioreactors, as well as for genetic improvement of livestock and the establishment of experimental models of diseases. However, there are not many practical applications of transgenic animals at present. In addition to the medical model of transgenic mice modified by a single gene, the research on the production of drug proteins by mammary gland bioreactor of transgenic animals has been going on for a long time, which has been more than 10 years. But at present, only two drugs in the world have entered the phase III clinical trial, and 5 ~ 6 drugs have entered the phase II clinical trial. However, transgenic livestock strains whose agronomic traits have been improved and can be used for livestock production have not yet been born. The low production efficiency of transgenic animals, the high cost and regulation failure caused by the difficulty of fixed-point integration, and the separation of genetic characters of sexually propagated offspring of transgenic animals, which are difficult to maintain the excellent victory of ancestors, are the main reasons that restrict the practical process of transgenic animals today.
The success of somatic cell cloning has set off a new revolution for the production of transgenic animals, and animal somatic cell cloning technology provides a technical possibility for rapidly amplifying the germplasm innovation effect produced by transgenic animals. Using simple somatic cell transfection technology to transfer the target gene can avoid the difficulty and inefficiency of livestock germ cells. At the same time, the transgenic cell line can be used for pre-inspection of transgenic integration and gender pre-selection under laboratory conditions. Before nuclear transfer, the fusion gene of target exogenous gene and marker gene (such as LagZ gene and neomycin antibiotic gene) was introduced into cultured somatic cells, and then transgenic positive cells and their clones were screened through the expression of marker genes, and then the nucleus of the positive cells was transplanted into enucleated oocytes. Theoretically, the final animal should be a 100% positive transgenic animal. By this method, Schnieke et al. (Bio Report, 1997) have successfully obtained 6 transgenic sheep, of which 3 have human coagulation factor IX gene and marker gene (neomycin resistance gene), and 3 have marker gene, and the integration rate of target foreign gene is as high as 50%. Cibelli (Science, 1997) also obtained three transgenic cows by nuclear transfer, which confirmed the effectiveness of this method. It can be seen that one of the most important application directions of animal cloning technology today is to develop transgenic cloned animals with high added value.
Embryonic stem cells are totipotent stem cells and have the potential to form all adult cell types. Scientists have been trying to induce various stem cells to differentiate into specific tissue types to replace those damaged tissues in the body, such as implanting insulin-producing cells into diabetic patients. Scientists have been able to transform pig es cells into beating cardiac myocytes, human ES cells into nerve cells and mesenchymal cells, and mouse ES cells into endodermal cells. These results open the way for cell and tissue replacement therapy. At present, scientists have successfully isolated human es cells (Thomson et al. 1998, Science), and somatic cell cloning technology provides the possibility for producing patients' own ES cells. The patient's somatic cells were transplanted into enucleated oocytes to form recombinant embryos, which were cultured into blastocysts in vitro. Then es cells were isolated from blastocysts and differentiated into specific cell types (such as nerve cells, muscle cells and blood cells) for replacement therapy. The ultimate goal of this nuclear transplantation method is to treat stem cells, not to obtain cloned individuals, which scientists call "therapeutic cloning".
The application of cloning technology in basic research is also very significant, which provides a tool for studying the mechanisms of gamete and embryogenesis, cell and tissue differentiation, gene expression regulation, nuclear-cytoplasmic interaction and so on.
Problems in verb (verb abbreviation) cloning technology
Although cloning technology has broad application prospects, it is still far from industrialization. As a new research field, cloning technology is not mature in theory and technology. Theoretically, the mechanism of reprogramming genetic material through differentiated somatic cell cloning (the process in which all or most genes in the nucleus are turned off and cells regain totipotency) is still unclear. Whether cloned animals will remember the age of donor cells, whether the continuous offspring of cloned animals will accumulate mutant genes, and the genetic role played by cytoplasmic mitochondria in the cloning process have not been solved.
In practice, the success rate of cloning animals is still very low. In the experiment of cultivating Dolly, Wilmut's research team fused 277 eggs with the transplanted nucleus, and only one live sheep Dolly was obtained, with a success rate of only 0.36%. At the same time, the cloning success rates of embryonic fibroblasts and embryonic cells were only 65,438 0.7% and 65,438 0.5%, respectively. 36866.88868688666
In addition, some born individuals show physiological or immune defects. Taking cloned cattle as an example, many cloned cattle cultivated in Japan, France and other countries died within two months after birth; By February 2000, 12 1 somatic cloned cattle had been born in Japan, but only 64 cows survived. The results showed that the placental function of some calves was not perfect, and the oxygen content and growth factor concentration in blood were lower than the normal level. The thymus, spleen and lymph glands of some calves are abnormal; Fetuses of cloned animals generally tend to develop faster than ordinary animals, which may be the cause of death.
Even Dolly, who developed normally, was found to have signs of premature aging. The end of the chromosome is called telomere, which determines the number of times a cell can divide: every time it divides, the telomere will be shortened, and when the telomere is exhausted, the cell will lose its ability to divide. 1998, scientists found that Dolly's cell telomeres are shorter than those of normal people, that is, its cells are in a more aging state. At that time, it was thought that this might be caused by cloning "Dolly" with adult sheep cells and making its cells bear the imprint of adult cells, but this explanation has been challenged at present. Lanza et al. cloned six calves from cultured aging cells. After 5 ~ 10 months of birth, it was found that the telomeres of these cloned calves were longer than those of ordinary calves of the same age, and some of them were even longer than those of ordinary newborn calves. At present, it is not clear why this phenomenon is different from Dolly's. However, this experiment shows that in some cases, the cloning process can change the molecular clock of mature cells and make them "rejuvenate". The effect of this change on the life span of cloned animals needs further observation.
In addition to the above theoretical and technical obstacles, the ethical impact of cloning technology (especially its application in human embryos) and the strong public reaction to it also limit its application. However, the development of cloning technology in recent years shows that most countries in the world are unwilling to lag behind, and no one has given up the research on cloning technology. At this point, the attitude of the British government is very representative. Less than 1 month after the end of February 1997, the British Science and Technology Council published a special report on cloning technology, indicating that the British government would reconsider this decision and think it unwise to ban this research blindly. The key is to establish certain norms and use them to benefit mankind.
A bacterium can split in two after about 20 minutes; A grape branch cut into ten segments may become ten grapes; Cactus is cut into several pieces, and each piece takes root when it hits the ground; A strawberry can grow hundreds of strawberry seedlings a year by its creeping stolons ... all these are the reproduction of offspring by an organism by splitting itself in two or expanding a small part of itself. This reproduction is called asexual reproduction. The English name of asexual reproduction is "Clone", transliterated as "Clone". In fact, the word "clone" in English comes from the Greek word "clone". Today, the meaning of "cloning" is not only "asexual reproduction", but also refers to a group of individuals who have undergone asexual reproduction from an ancestor. This group of asexual offspring from an ancestor is also called "asexual cloning", or cloning for short.
Many animals in nature, under normal circumstances, rely on male cells (sperm) produced by their fathers and female cells (eggs) produced by their mothers to fuse (fertilize) into fertilized eggs (zygotes), and then the fertilized eggs develop into embryos through a series of cell division, and finally form new individuals. This kind of reproduction mode, which relies on the sex cells provided by both parents and produces offspring through the fusion of bisexual cells, is called sexual reproduction. However, if we divide the embryo into two parts, four parts and eight parts through surgery ... finally, an embryo grows into two, four and eight ... organisms through special methods. These creatures are cloned individuals, and these two, four, eight ... individuals are called clones (also known as clones).
It can be said that Wu Cheng'en, a great writer in China in the Ming Dynasty, once described the idea of cloning wonderfully-the Monkey King often pulled out a handful of monkey hair at a critical moment and turned it into a large group of monkeys, and the monkey hair turned into a monkey is a cloned monkey.
1In the spring of 979, scientists from Wuhan Institute of Hydrobiology, Chinese Academy of Sciences artificially cultured the cells in the blastocyst stage of crucian carp. After 59 generations of continuous subculture for 385 days, the nucleus was sucked out of the cultured cells by a glass tube with a diameter of about 65438 00 microns under a microscope. At the same time, the nucleus of crucian carp eggs was removed to prepare the eggs for accepting blastocyst nuclei. After everything is ready, the nucleus sucked out of the glass tube is moved into the empty position of crucian carp eggs, and most of the blastocyst nuclei under artificial culture die prematurely. Of the 189 eggs exchanged by nuclear exchange, only two hatched fry, and finally only one young fish survived the difficulties. After more than 80 days of culture, it grew into a crucian carp with a body length of 8 cm. This kind of crucian carp has not been combined with male and female cells, but only replaced the nucleus of a blastocyst with an egg cell, and the egg cell is actually produced by the egg after nuclear replacement, so it is also a cloned fish.
Before the emergence of cloned crucian carp, scientists from Oxford University in England had conducted cloning experiments with a Xenopus laevis (Xenopus laevis) in 1960 and 1962. The experimental method is to irradiate Xenopus laevis eggs with ultraviolet rays to destroy their nuclei, and then take out the nuclei from the intestinal epithelial cells, liver cells and kidney cells of Xenopus laevis tadpoles through superb surgery, and accurately put the nuclei of these cells into the eggs whose nuclei have been destroyed by ultraviolet rays. After careful care, some of these nuclear exchange eggs finally grew up alive and kicking Xenopus laevis, which was not produced by the combination of sperm cells and egg cells, so it was also a cloned Xenopus laevis.
Tong Dizhou, a famous biologist in China, successfully conducted the cloning experiment of Rana nigromaculata in 1978. He transplanted the red cell nucleus of Rana nigromaculata into the eggs of Rana nigromaculata which had been enucleated in advance, and the eggs after enucleation eventually grew into tadpoles that could swim freely in the water.
With the maturity of fish nuclear exchange technology and the success of amphibian nuclear exchange, a group of scientists engaged in seed cultivation are excited. Since the nucleus of crucian carp blastocysts can replace the nucleus of crucian carp eggs to obtain cloned fish, can the nuclear exchange of heterogeneous fish get new hybrid fish? Scientists in China first proposed and solved this problem, that is, the research institute that successfully cultivated cloned crucian carp managed to replace the nucleus of crucian carp embryo cells with the nucleus of crucian carp egg cells. The nucleus of carp and the cytoplasm of crucian carp eggs can coexist peacefully and begin a process similar to the division and development of fertilized eggs. Finally, a kind of "crucian carp" with a beard grows very fast, just like carp, but its lateral scales and spines are the same as those of crucian carp, and the taste of fish is no less than that of crucian carp. The emergence of this new species of artificially cloned fish has opened up a new way for fish breeding.
The pursuit of science is endless, and the success of fish and amphibian cloning naturally makes scientists turn their attention to mammals. Scientists in the United States and Switzerland took the lead in taking out the nucleus from the embryonic cells of gray mice and replacing the nucleus of fertilized eggs of black mice with this nucleus. In fact, the fertilized egg of this black mouse just entered the egg cell and removed the sperm nucleus together with the egg nucleus. After transplanting the embryo nucleus of squirrel into the enucleated fertilized egg of a black mouse, it was artificially cultured in a test tube for four days and then implanted into the uterus of a white mouse. After hundreds of gray, black and white operations, the white mouse finally gave birth to three little squirrels.
The British magazine Nature published on February 27th last year published the research results of wilmot and others of Roslin Institute in Edinburgh: After 247 failures, they got a cloned ewe named Dolly in July the year before last.
How did Dolly sheep "create"? Wilmot and other scholars first injected gonadotropin into Scottish black-faced sheep to induce ovulation. After getting the egg, they immediately took out the nucleus from the egg cell with a very thin straw. At the same time, they took out the nucleus from the mammary gland cells of a six-year-old ewe named Fendosit, who was pregnant for three months, and immediately sent it to the egg cells of the enucleated Scottish black-faced sheep. After the operation, they used electric pulses with the same frequency to stimulate egg exchange. Let the cytoplasm of Scottish black-faced sheep and the nucleus of breast cells of FenDorset ewe coordinate with each other, let this "assembled" cell undergo the process of division and development like a fertilized egg in a test tube, form an embryo, and then skillfully implant the embryo into the uterus of another ewe. In July last year, the ewe that "nursed" the embryo in vitro finally gave birth to Dolly, a lamb. Dolly is not the product of fertilization between ewe eggs and ram sperm cells, but the result of the step-by-step development of "nuclear-egg exchange", so it is "cloned sheep"
The birth of "cloned sheep" shocked all countries in the world. Its commendable feature is that it is the nucleus of somatic cells, not the nucleus of embryos. This result proves that the so-called highly differentiated cells that perform special functions and have specific forms in animals have the potential to develop into complete individuals like fertilized eggs. In other words, animal cells are totipotent like plant cells.
Cloning technology will bring great benefits to mankind. For example, the ewe bred by British PPL company contains a- 1 antitrypsin for treating emphysema. The price of this goat's milk is $6,000 a liter. A ewe is like a pharmaceutical factory. What is the most effective and convenient way to breed this kind of sheep? The best way is to "clone". Similarly, the Dutch PHP company has bred cows that can secrete human lactoferrin, and the Israeli LAS company has bred sheep that can produce serum albumin. How can these high value-added livestock breed effectively? The answer is of course "cloning".
A mare with a donkey can get an animal with particularly strong heterosis-mule. Mules can't reproduce, so how can excellent mules expand their reproduction? The best way is also "cloning". The giant panda in China is a national treasure, but its natural mating success rate is low and it is on the verge of extinction. How to save such rare animals? "Cloning" provides a practical way for human beings.
Cloning animals also plays an important role in studying cancer biology, immunology and human life span.
Undeniably, the appearance of "cloned sheep" has also aroused many people's interest in "human cloning". For example, some people are considering whether they can clone an embryo from their own cells and freeze it before it takes shape. One day in the future, when something goes wrong with one of our own organs, we can take this organ out of the embryo for culture and then replace our diseased organs, which is to provide ourselves with "accessories" through cloning.
The discussion about "human cloning" reminds people that scientific and technological progress is a mixed March. The more science and technology develop, the wider and deeper it penetrates into society, and the more likely it is to cause many related ethical, moral and legal problems. I want to end this article with a sentence from J.D. Watson, a famous molecular biologist who won the Nobel Prize: "It can be expected that many biologists, especially those engaged in asexual reproduction research, will seriously consider its significance and start scientific discussions to educate people all over the world."