Cloning is an exact reproduction of an object any required number of times. Objects obtained as a result of cloning (each separately and their entire collection) are called a clone.
Natural cloning of animals and plants often occurs as a result of asexual and vegetative reproduction, as well as as a result of ameiotic parthenogenesis.
Artificial cloning of animals and plants is a new type of human activity that emerged in the late XX – early XXI century, consisting in the reproduction of old and the creation of new biological organisms associated with the study of the genome, involving interference in its structure, aimed at solving many practical problems (except for scientific ).
The term cloning” was originally used in microbiology and selection, after – in genetics, due to the success of which they came into general use. It should be added that cinema and literature also contributed to their popularization.
One should bear in mind that accurate reproduction of an animal or plant, both with natural and artificial cloning, is impossible. In any case, the new organism will differ from the maternal one due to somatic mutations, epigenetic changes in hereditary material, the effect of the environment on the phenotype, and random deviations arising during ontogenesis.
The creation of animals and plants with the given qualities has always been extremely tempting because it meant creating the most unique and essential organisms, resistant to diseases, climatic conditions, giving sufficient offspring, the required amount of meat, milk, fruits, vegetables and other products.
The use of cloning technology implies a unique opportunity to obtain phenotypically and genetically identical organisms that can be used to solve various theoretical and applied problems facing biomedicine and agriculture. In particular, the use of cloning could contribute to the study of the problem of totipotency of differentiated cells, the development and aging of organisms, and malignant degeneration of cells. Thanks to cloning technology, the emergence of accelerated genetic selection and replication of animals with exceptional production indicators is expected. In combination with transgenesis, animal cloning opens up additional opportunities for the production of valuable biologically active proteins for the treatment of various diseases of animals and humans. Animal cloning may allow testing of medicines on identical organisms.
Plant cloning is carried out by regenerating a whole plant from callus by changing the proportional ratio of cytokinins and auxins in the nutrient medium. To obtain primary callus, you can use any plant cells and tissues (except those in a premortal state) due to the fact that plant cells are capable of dedifferentiation at certain concentrations of phytohormones in the nutrient medium. But more often meristem cells are used for this purpose due to their low degree of differentiation. Auxin (for cell dedifferentiation) and cytokinin (for the induction of cell division) are necessarily included in the nutrient medium for callus formation. After receiving a callus culture, the callus can be divided and each part used to regenerate whole plants. Since callus is a formless undifferentiated cell mass, for plant regeneration it is necessary to induce morphogenesis by changing the concentrations of phytohormones in the environment. Cloning of plants allows obtaining virus-free planting material (using the apical meristem as a source of cells), rapid reproduction of plants on a large scale (including rare and endangered ones), cloning from anthers and subsequent restoration of diploidy allows to obtain plants homozygous for all genes that can be used in further selection. It is also possible to cultivate protoplasts of plants on artificial nutrient media, from which in some cases it is possible to regenerate whole plants (protoplasts are convenient for transgenesis due to their lack of a cell wall and the possibility of fusion with other cells).
In its early days:
The first successful experiments on animal cloning were carried out in the 1960s by the English embryologist J. Gurdon in experiments on the clawed frog. Tadpole intestinal cell nuclei were used for transplantation. The experiments were criticized since primary germ cells could be preserved in the intestines of the tadpoles. In 1970, experiments were carried out in which the replacement of the egg nucleus with a genetically labeled nucleus from the somatic cell of an adult frog led to the appearance of tadpoles and adult frogs. This showed that the technique of nuclear transplantation from somatic cells of adult organisms into enucleated (nucleus-deprived) oocytes makes it possible to obtain genetic copies of an organism that served as a donor of differentiated cell nuclei. The result of the experiment became the basis for the conclusion about the reversibility of embryonic differentiation of the genome, at least in amphibians.
Cloning of mammals is possible by means of experimental manipulations with oocytes and nuclei of animal somatic cells in vitro and in vivo. Cloning of adult animals is achieved as a result of the transfer of a nucleus from a differentiated cell to an unfertilized egg, from which its own nucleus (enucleated egg) has been removed, followed by transplantation of the reconstructed egg into the foster mother’s oviduct. However, for a long time, all attempts to apply the method described above for cloning mammals were unsuccessful. One of the first successful cloning of a mammal (house mouse) was carried out by Soviet researchers in 1987. They used the electroporation method for the fusion of an enucleated zygote and a mouse embryonic cell with a nucleus.
A significant contribution to solving this problem was made by a Scottish team of researchers from the Rosslyn Institute and PPL Therapeuticus (Scotland) under the leadership of Ian Wilmut. In 1996, their publications appeared on the successful birth of lambs as a result of transplantation of nuclei obtained from fibroblasts of a sheep fetus into enucleated oocytes. In the final form, the problem of animal cloning was solved by Wilmuth’s group in 1996, when a sheep named Dolly was born – the first mammal obtained from the nucleus of an adult somatic cell: the oocyte’s own nucleus was replaced with a cell nucleus from the culture of mammary epithelial cells of an adult lactating sheep. Subsequently, successful experiments were carried out on the cloning of various mammals using nuclei taken from adult somatic cells of animals (mouse, goat, pig, cow), as well as taken from dead animals frozen for several years. The emergence of animal cloning technology not only aroused great scientific interest but also attracted the attention of big business in many countries.
In 2004, the Americans began commercial cloning of cats, and in April 2008, South Korean customs officers began training seven puppies cloned from the somatic cells of the best Korean wanted dog, the Canadian Labrador Retriever. According to South Korean scientists, 90% of cloned puppies will meet the requirements for working at customs, while only less than 30% of ordinary puppies pass aptitude tests.
In China, the BGI firm is already producing animal cloning for medical research on an industrial scale. It is assumed that a similar technique will be used in the future to grow spare organs in pigs for human transplantation.
In 2009, a work was published in which it was shown for the first time that induced pluripotent stem cells (iPSCs) can give rise to a full-fledged organism, including its germline cells (using the tetraploid complementation method,). iPS derived from mouse skin fibroblasts by transformation with a retroviral vector yielded in some percentage of cases healthy adult mice that were able to reproduce normally. Thus, for the first time, cloned animals were obtained without the admixture of the genetic material of the oocytes (with the standard cloning procedure, mitochondrial DNA is transferred to the offspring from the recipient’s oocyte).
Cloning can be used to recreate natural populations of extinct animals. Despite the presence of certain problems and difficulties, the first results in this direction are already available.
In Spain, in 2003, a cloned cub of the extinct subspecies of the Pyrenean ibex bucardo (Capra pyrenaica) was born. The cloning was reported in the January issue of Theriogenology magazine.
This subspecies of the Pyrenean goats completely disappeared by 2000 (the reasons for the extinction are not exactly known). The last member of the species, a female named Celia, died in 2000. But before that (in 1999) Jose Folch of the Research Center for Agriculture and Technology of Aragon (CITA) took several skin cells from Celia for analysis and storage in liquid nitrogen. This genetic material was used in the first attempt to clone an extinct subspecies.
Experimenters transferred bucardo’s DNA into domestic goat eggs, devoid of their own genetic material. The resulting embryos were planted in surrogate mothers – females of other subspecies of the Spanish goat or hybrid species obtained by crossing domestic and wild goats. Thus, 439 embryos were created, 57 of which were implanted into the surrogate uterus. Only seven operations ended in pregnancy and only one goat eventually gave birth to a female Bucardo, who died seven minutes after birth from respiratory problems.
Despite the failed cloning and the death of the cloned goat, many scientists believe that such an approach may be the only way to save the endangered species. This gives scientists hope that endangered and recently extinct species can be resurrected using frozen tissue.
In 2004, a pair of bantengs (wild bulls that lived in Southeast Asia) were born, cloned from the cells of animals that died more than 20 years ago. The two bantengs were cloned from San Diego’s unique “frozen zoo”, created before humans even realized cloning was possible. The American company said it used cells from animals that died in 1980 without leaving any offspring.
Bantengs were cloned by transferring their genetic material into the empty eggs of ordinary domestic cows; only two survived out of 16 embryos.
The Imperial Woodpecker was last seen in Mexico in 1958. Since then, bird watchers have been trying to find traces of this population, but to no avail. About ten years ago there were even rumors that the bird was still living on the planet, but they were not confirmed.
However, there are stuffed birds in the museums. A researcher at the Darwin Museum, Igor Fadeev, believes that if the operation to isolate DNA is carried out with all the stuffed animals that are in different countries of the world, then the woodpecker can be resurrected. In various museums around the world, today there are only ten stuffed imperial woodpeckers.
If the project is successful, then in the near future, the imperial woodpecker may reappear on our planet. The State Darwin Museum is confident that the latest methods of molecular biology make it possible to isolate and reproduce the DNA of these birds.
In June 2006, Dutch scientists discovered the well-preserved remains of a dodo, a flightless bird that has recently become extinct (in the 17th century), on the island of Mauritius. Previously, science did not have the remains of a bird. But now there is a certain hope for the “resurrection” of this representative of birds
Plans to clone extinct giant birds have been challenged by research by scientists at Oxford University. By isolating sections of DNA from the remains of extinct birds, scientists found that their genetic material was so destroyed that modern technology does not allow full-fledged cloning. The purpose of scientific work was to revive the extinct several centuries ago, the New Zealand ostrich moa, as well as the Madagascar epyornis (elephant bird).
DNA samples were taken from tissue fragments preserved in museums. However, scientists were unable to obtain a DNA strand long enough to carry out cloning. Nevertheless, some scientists believe that in the coming years, the technology will be developed to restore the lost parts of DNA by stitching there “patches” from the DNA of closely related species.
Later, the research team of Michael Bunce of Murdoch University (Australia) developed an effective method for extracting DNA from the shell of fossil eggs, which has been shown to be effective on the shell of moa and epyornis eggs up to 19,000 years old, which makes plans to clone giant fossil birds more realistic.
Laboratory George Church from Harvard University (USA) in mid-October 2014 announced the start of a project to “resurrect” mammoths. The chances for the resurrection of mammoths appeared thanks to the appearance of the revolutionary CRISPR / CAS genome rewriting technology, which allows you to point-wise change and delete arbitrary genes in mammalian DNA (in 2012). Using this technique, Church and his colleagues were able to successfully insert genes into the genome of elephant skin cells believed to be responsible for the typical mammoth traits – small ears, thick subcutaneous fat, long hair, and brown color. The cells survived this transformation and now scientists are thinking about how they can be turned into the real skin tissue.
In March 2015, it was announced that American geneticists were able to successfully transplant part of the mammoth genes extracted from DNA fragments of ice age giants into the genome of a cell of an ordinary African elephant and reproduce them. Thus, geneticists took the first step towards the resurrection of the mammoth or the creation of a mammoth elephant.
In May 2015, the journal Current Biology published an article on the deciphering of the genome of two mammoths. Perhaps the new data will find application in the cloning of mammoths, but so far experts cannot do without the eggs of modern elephants.
There is commercial pet cloning. First, deceased domestic cats and dogs are cloned. So, by 2020, Barbra Streisand twice cloned her dog Samantha, who died in 2017. Cloning is expensive. In 2015, in South Korea, they charged about 100 thousand dollars for cloning a dog, while in the United States it was half that.
In China, cloning a dog in 2020 cost 54 thousand dollars. The Chinese authorities cloned the police dog Kunxun, which they began to train in 2019. Second, racehorses are cloned. In Texas, one of the companies by 2020 created 25 clones of the mare Aiken Kura (she won races), one of which was sold for 800 thousand dollars.
They are also cloned for the production of expensive meat. In 2015, near Tianjin, they began to build a factory for the production of premium beef from cloned cows. In 2018, the US Food and Drug Administration authorized the sale of meat and milk from cloned animals.