“Clone” is transliterated from the English “clone” and has three different levels of meaning in the field of biology.
1. At the molecular level, cloning generally refers to DNA cloning (also called molecular cloning). It means that a specific DNA fragment is inserted into a vector (such as plasmid and virus, etc.) through recombinant DNA technology, and then replicates itself in the host cell to obtain a large number of identical "populations" of the DNA fragment.
2． At the cellular level, a clone is essentially a cell population formed by the division of a single ancestral cell. Each cell is genetically identical. For example, a cell group with the same genetic background formed by dividing a cell for several generations in culture medium in vitro 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 responses. All plasma cells that produce a specific antibody are divided from one B cell, and such a plasma cell population is also a cell clone. Cell cloning is a low-level reproductive method - asexual reproduction, that is, without sexual union, the offspring have the same genetics as the parents. The lower the level of biological evolution, the more likely it is to adopt this method of reproduction.
3. At the individual level, a clone refers to a population of two or more individuals with identical genotypes. For example, two identical twins are one clone! Because they come from the same egg cell, they have exactly the same genetic background. By this definition, "Dolly" cannot be said to be a clone! Because "Dolly" is just a lonely one. Only when those British embryologists can transplant two or more identical cell nuclei into two or more identical enucleated egg cells and obtain two or more "Dolly" with exactly the same genetic background can the term cloning be used to describe it. So in the sensational paper published in Nature in February 1997, the author did not describe "Dolly" as a clone.
In addition, cloning can also be used as a verb, which means the process of obtaining the above-mentioned DNA, cells or individual groups.
2. Cloning technology
1. DNA cloning
There are many methods for DNA cloning. The basic process is shown in the figure below (not to scale).
It can be seen that the DNA obtained in this way can be used in biological research. Many aspects include the analysis and processing of specific DNA base sequences, as well as the mass production of valuable proteins in the biotechnology industry.
2． Cloning of biological individuals
(1) Cloning of plant individuals
In the 1950s, botanists used carrots as model materials to study whether genetic material was lost in differentiated plant cells. Problem, they were surprised to find that a complete plant could develop from a single highly differentiated carrot cell! From this, they believed that plant cells are totipotent. The genetic background of the carrot population developed from more than two somatic cells in one carrot is exactly the same, so it is a clone. Such a plant cloning process is a complete asexual reproduction process!
(2) Cloning of individual animals
① The birth of “Dolly”
1997 On February 27, Ian Wilmot's scientific research team at the Roslin Institute in Edinburgh, UK, announced to the world that the world's first cloned sheep, "Dolly", was born. This news immediately caused a sensation around the world. .
The creation of "Dolly" is related to three ewes. One is a three-month-old Finnish Dorset ewe, and the two are Scottish black-faced ewes. The Finnish Dorset ewe provided the complete set of genetic information, that is, the nucleus (called the donor); a Scottish Blackface ewe provided the egg cell without a nucleus; and another Scottish Blackface ewe provided the development of the sheep embryo. The environment - the uterus, is the "birth" mother of "Dolly" sheep.
The entire cloning process is briefly described as follows:
Mammary gland cells were taken out from the mammary glands of Dorset ewe sheep in Finland and put into low-concentration nutrient culture medium. The cells gradually stopped dividing. This cell is called It is a donor cell; a Scottish black-faced ewe is injected with gonadotropin to cause it to ovulate, the unfertilized egg cell is taken out, and its nucleus is immediately removed, leaving a nucleus-free egg cell. This cell is called the recipient. cells; the method of electric pulse is used to fuse the donor cell and the recipient cell, and finally a fused cell is formed. Since the electric pulse can also produce a series of reactions similar to the natural fertilization process, the fused cell can also behave like a fertilized egg. The cells divide and differentiate in the same way to form embryonic cells; the embryonic cells are transferred to the uterus of another Scottish black-faced ewe, and the embryonic cells further differentiate and develop, finally forming a lamb. Dolly lambs are born with the exact same appearance as Dorset ewes.
A year later, another group of scientists reported that more than 20 cells were obtained by transplanting the nuclei of mouse cumulus cells (highly differentiated cells surrounding the periphery of oocytes) into oocytes with their nuclei removed. Only fully developed mice. If there is only one "Dolly" and it is not enough to be called a cloned sheep, these mice are truly cloned mice.
② The basic process of cloning mice through nuclear transfer
In this experiment, cumulus cells were obtained through the following process: through several consecutive injections of chorionic gonadotropin, Inducing female mice into a state of high egg production. Complexes of cumulus cells and oocytes were then collected from the oviducts of female mice. Cumulus cells were dispersed by hyaluronic acid treatment. Select cumulus cells with a diameter of 10-12 microns as the nucleus donor (previous experiments have shown that if the nuclei of cumulus cells with smaller or larger diameters are used, the oocytes that have undergone nuclear transplantation will rarely develop to the 8-cell stage. ). The selected cumulus cells were kept in a certain solution environment and the nuclei were transplanted within 3 hours (different from this, the breast cells used as nucleus donors when obtaining "Dolly" were first passaged in the culture medium for 3 hours. -6 times)
Oocytes (generally in meiotic metaphase II) were collected from female mice of different species by a method similar to that described above. Carefully remove the nucleus of the oocyte using a thin tube with a diameter of approximately 7 microns under a microscope, and try not to remove the cytoplasm. Also carefully remove the nucleus of the cumulus cells and remove as much cytoplasm as possible (by moving the removed nucleus back and forth in the glass tube several times to remove a small amount of cytoplasm). Inject directly into the oocyte from which the nucleus has been removed within 5 minutes after the nucleus is removed. The oocytes that have undergone nuclear transfer are first placed in a special solution for 1-6 hours, and then divalent strontium ions (Sr2+) and cytostatin B are added. The former activates the oocyte, and the latter inhibits the formation of polar bodies and the elimination of chromosomes. The treated oocytes are then taken out and placed in a special solution without strontium and cytostatin B to cause cell division to form embryos.
Embryos of different stages (from the 2-cell stage to the blastocyst stage) were implanted into the fallopian tubes or uterus of pseudopregnant female mice that had been mated with ligated male mice a few days ago. Fully developed fetal mice are surgically removed after approximately 19 days.
At present, animals cloned by embryonic cell nuclear transfer include mice, rabbits, goats, sheep, pigs, cows and monkeys. In China, in addition to monkeys, other animals are cloned, and goats can also be cloned by continuous nuclear transfer. This technology goes further than embryo segmentation technology and will clone more animals. Because the more times the embryo is divided, the fewer cells each share, and the worse the ability of the individual to develop. There is only one animal cloned by somatic cell nuclear transfer, and that is Dolly the sheep.
3. The gospel of cloning technology
1. Cloning technology and genetic breeding
In agriculture, people have used "cloning" technology to cultivate a large number of high-quality, high-yielding varieties that are resistant to drought, lodging, and pests and diseases, which has greatly increased grain yields. In this regard, our country has entered the forefront of the world's most advanced countries.
2． Cloning technology and the protection of endangered organisms
Cloning technology is a boon to the protection of species, especially rare and endangered species, and has great application prospects. From a biological perspective, this is also one of the most valuable aspects of cloning technology.
3. Cloning Technology and Medicine
In modern times, doctors can perform transplant operations on almost all human organs and tissues. But in terms of science and technology, rejection in organ transplantation is still the most troublesome thing. The reason for rejection is poor tissue compatibility due to tissue mismatch. If the organs of a "cloned human" are provided to an "original human" for organ transplantation, there will be absolutely no worries about rejection, because the genes and tissues of the two are matched. The question is, is it humane to use "human clones" as organ donors? Is it legal? Does it make financial sense?
Cloning technology can also be used to reproduce valuable genes in large quantities. For example, in medicine, people use "cloning" technology to produce insulin to treat diabetes and growth hormone to make patients with dwarfism grow taller again. And streptozolin, which can resist various viral infections, etc.