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) by recombinant DNA technology, and then replicated in the host cell to obtain a large number of identical "groups" of the DNA fragment.
2. At the cellular level, cloning is essentially a cell group formed by the division of a single * * * same ancestor cell. Each of these cells has the same gene. For example, a cell group with the same genetic background formed by dividing a cell for several generations in vitro culture medium 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 reaction. All plasma cells that produce a specific antibody are formed by the division of a B cell, and such a plasma cell population is also a cell clone. Cell cloning is a low-level reproductive mode-asexual reproduction, that is, offspring and parents have the same heredity without sexual union. The lower the level of biological evolution, the more likely it is to adopt this way 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" can't be said to be a clone! Because "Dolly" is just a lonely one. Only when those British embryologists can transplant more than two identical nuclei into more than two identical enucleated eggs and get more than two "Dolly" with identical genetic background can they use the word cloning to describe it. So in that sensational paper published in Nature in February 1997, the author did not say "Dolly" was a clone.
in addition, cloning can also be used as a verb, which means the process of obtaining the DNA, cells or individual groups mentioned above.
ii. cloning technology
1. DNA cloning
There are various methods for DNA cloning at present, and the basic process is shown in the following figure (not to scale)
It can be seen that the obtained DNA 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, etc.
2. Cloning of biological individuals
(1) Cloning of plant individuals
In 195s, botanists used carrots as model materials to study whether genetic materials were lost in differentiated plant cells, and they were surprised to find that a complete plant could be developed from a single highly differentiated carrot cell
! Therefore, they think that plant cells are totipotent. 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 such a plant is a complete asexual reproduction process!
(2) Cloning of Individual Animals
① Birth of Dolly
On February 27th, 1997, 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 Finnish Dorset ewe who is pregnant for three months, and the other is a Scottish black-faced ewe. The Dorset ewe in Finland provided a complete set of genetic information, that is, provided the nucleus (called the donor); A Scottish black-faced ewe provides egg cells without nucleus; Another Scottish black-faced ewe provides the development environment of sheep embryos-uterus, which is the "biological" mother of Dolly sheep. 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 culture medium, and the cells gradually stopped dividing, which was called donor cells; Inject gonadotropin into a Scottish black-faced ewe to induce it to ovulate, take out the unfertilized egg cell, and immediately remove its nucleus, leaving a seedless egg cell, which is called the recipient cell; Using the method of electric pulse, the donor cell and the recipient cell are fused, and finally a fused cell is formed. Because the electric pulse can also produce a series of reactions similar to the natural fertilization process, the fused cell can also divide and differentiate like a fertilized egg, thus forming an embryonic cell. The embryonic cells were transferred to the uterus of another Scottish black-faced ewe, and the embryonic cells further differentiated and developed, and finally a little sheep was formed. Dolly lamb born has exactly the same appearance as Dorset ewe.
a year later, another group of scientists reported that more than 2 fully developed mice were obtained by transplanting the nucleus of mouse cumulus cells (highly differentiated cells around 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 < P > are veritable cloned mice.
② Basic process of cloning mice by nuclear transfer
In this experiment, cumulus cells were obtained by the following process: by injecting chorionic gonadotropin several times in a row, female mice were induced into a state of high egg production. Then the complex of cumulus cells and oocytes was collected from the fallopian tubes of female rats. The cumulus cells were dispersed by hyaluronic acid treatment. Cumulus cells with a diameter of 1-12 microns were selected as nuclear donors (previous experiments showed that if the nuclei of cumulus cells with smaller or larger diameters were used, oocytes after nuclear transfer rarely developed to 8-cell stage). The selected cumulus cells were kept in a certain solution environment, and the nuclear transfer was carried out within 3 hours (unlike this, the breast cells used as nuclear donors were first propagated in the culture medium for 3-6 times when Dolly was obtained)
Oocytes (usually in the metaphase of meiosis II) were collected from different female mice by similar methods as 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 carefully remove the nucleus of cumulus cells, and try to remove the cytoplasm (by reciprocating the removed nucleus in the glass tube several times to remove a small amount of cytoplasm). Within 5 minutes after the nucleus is taken out, it is directly injected into the oocytes from which the nucleus has been removed. The oocytes with nuclear transfer are put in a special solution for 1-6 hours, and then bivalent strontium ions (Sr2+) and cytochalasin B are added. The former activates oocytes, while the latter inhibits the formation of polar bodies and the elimination of chromosomes. Then the treated oocytes are taken out and placed in a special solution without strontium and cytochalasin B to divide the cells into embryos.
Embryos in different stages (from 2-cell stage to blastocyst stage) were implanted into the fallopian tubes or uterus of pseudo-pregnant female rats that had been mated with male rats a few days ago for development. The fully developed fetal rats were removed by surgery after about 19 days.
At present, the animals cloned by embryo nuclear transfer include mice, rabbits, goats, sheep, pigs, cows and monkeys. In China, there are all cloned animals except monkeys, and goats can also be cloned by continuous nuclear transfer. This technology goes further than embryo segmentation technology, and more animals will be cloned. Because the more times the embryo is divided, the fewer cells there are in each part, and the worse the ability of the developed individual is. There is only one animal cloned by somatic cell nuclear transfer, which is Dolly sheep.
3. Gospel of cloning technology
1. Cloning technology and genetic breeding
In agriculture, people use "cloning" technology to cultivate a large number of high-quality and high-yield varieties with drought resistance, lodging resistance and disease and insect pest resistance, which greatly improves the grain yield. In this respect, China has entered the forefront of the world's most advanced.
2. Cloning technology and the protection of endangered species
Cloning technology is a gospel for the protection of 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 perform transplantation on almost all human organs and tissues. But as far as science and technology are concerned, rejection in organ transplantation is still the most headache. The reason of rejection is that tissue mismatch leads to poor compatibility. If the organs of the "cloned human" are provided to the "original human" for organ transplantation, there is absolutely no fear of rejection, because the genes and tissues of the two are matched. The question is, is it humane to use "human cloning" as an organ donor? Is it legal? Is the economy economical?
Cloning technology can also be used to multiply valuable genes in large quantities. For example, in medicine, it is through "cloning" technology that people produce insulin to treat diabetes, growth hormone to make dwarfism patients grow taller again, and drynarine to resist various virus infections, and so on.