The second is to cultivate a large number of excellent livestock, such as cattle, sheep and pigs with good meat quality, and also cultivate some cows with high milk production and rich nutrients needed by human body.
Third, it has great influence on medical treatment, such as finding out the pathogenic genes by molecular cloning technology and putting forward the molecular biological mechanism of diseases; Cloning a transgenic sheep whose milk contains drug protein for treating hemophilia can better meet the dietotherapy needs of hemophilia patients. Seeking a wider source of organ transplantation, introducing human organ tissues and immune system genes into animals to grow the needed human organs can reduce immune rejection and improve the success rate of transplantation.
Fourth, in order to protect the environment and endangered animals and plants, species are propagated through cloning technology.
The fifth is to provide more suitable animals for medical research and greatly improve the accuracy and safety of the experiment.
Sixth, a large number of crops can be planted in a short time, which can alleviate the food crisis.
Difficulties: First, the early research of cloning.
The word clone is a transliteration of the English word clone. As a noun, c 1one 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 first put forward by Hans Spearman in 1938. He called it a "strange experiment", that is, taking out the nucleus from a developing embryo (mature or immature embryo) 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 first achievement of mammalian embryo nuclear transfer research was obtained in 198 1 year-Karl Ilmen Ze and Peter Hope used mouse embryonic cells to culture normal mice. 1984, Steen Willadsen cloned a live-born sheep with immature embryo cells from sheep. Later, some people repeated his experimental method with various animals such as cattle, pigs, goats, rabbits and macaques. 1989, Willadson obtained the second generation of cloned cattle with continuous nuclear transfer. In 1994, Neil Fierst cloned a cow from a late embryo 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 single gene, the research on the production of drug protein by mammary gland bioreactor of transgenic animals has been carried out for a long time, and it has been 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 the cloning of Dolly from adult sheep cells, which made its cells bear the imprint of adult cells. However, this explanation is now questioned. Robert Lanza, a doctor in Massachusetts, USA, cloned cattle from cultured aging cells and got six calves. After 5 ~ 10 months of birth, it was found that the telomeres of these cloned cows were longer than those of ordinary calves of the same age, and some 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.