1, cell culture technology
Cell culture technology is the basic technology of cell engineering. The so-called cell culture is the technology of taking a small piece of a certain part of the organism and culturing it to make it grow and divide. Cell culture is also called tissue culture. In recent twenty years, some important theoretical research progress in cell biology, such as the revelation of cell totipotency, cell cycle and its regulation, research on canceration and cell aging, gene expression and regulation, etc. Are inseparable from cell culture technology.
In vitro cell culture is a medium for supplying nutrients needed by the whole animal and plant cells. In addition to rich nutrients, the culture medium generally contains some trace substances that stimulate cell growth and development. Generally, there are two kinds of culture media, solid and liquid, which can only be used after sterilization. In addition, temperature, light and oscillation frequency are also important conditions affecting culture.
The basic process of plant cell and tissue culture includes the following steps:
The first step is to select starting materials (explants) for culture from specific parts or tissues of healthy plants, such as roots, stems, leaves, flowers, fruits and pollen.
The second step is to disinfect the surface of the explant with some chemicals (sodium hypochlorite, mercury chloride and alcohol, etc.). ) establish the sterile culture system.
Thirdly, callus and organs are formed, and the callus differentiates into buds, which can be further induced to form small plants.
There are two ways of animal cell culture. One is called non-monolayer culture: that is, cells do not adhere to the wall during the culture process, and the conditions are more complicated and more difficult, but it is easy to obtain a large number of cultured cells at the same time. This method is usually used for the culture of lymphocytes, tumor cells and some transformed cells. Another culture method is monolayer culture: it is also called cell attachment, and the attached cells grow in monolayer, so this method is also called monolayer cell culture. Most mammalian cells must be cultured in this way.
Animal cells cannot be cultured in vitro. Taking human skin cell culture as an example, the main steps of animal cell culture are as follows:
The first step is to take a proper amount of tissue from healthy animals under aseptic conditions and cut it into small slices.
Step 2, add appropriate concentration of enzymes and auxiliary substances for digestion to disperse the cells.
Thirdly, after washing and purifying the dispersed cells, they were added to the culture medium at an appropriate concentration, cultured at 37℃ and subcultured at the right time.
In cell culture, we often use a word-cloning. The word clone is transliterated from English clone, which refers to asexual reproduction and cell population or biological population obtained through asexual reproduction. Cell cloning refers to the asexual reproduction line of cells. Natural cloning has long existed in nature. For example, identical twins are actually a kind of cloning.
In genetic engineering, there is another kind called molecular cloning, which was put forward by Cohen and others in 1973. Molecular cloning occurs at the level of DNA molecule, which refers to the DNA molecular cloning obtained by extracting a gene from a cell as a foreign gene, connecting it with a vector in vitro, and then introducing it into another recipient cell for autonomous replication.
2. Nuclear transfer technology
Because cloning is asexual reproduction, all members of the same clone have exactly the same genetic composition, which is conducive to faithfully maintaining the excellent characteristics of the original variety. People began to explore the artificial cloning of higher animals. There are two main methods of mammalian cloning: embryo segmentation and nuclear transfer. Among them, nuclear transplantation is a new technology with late development but great potential.
Nuclear transfer technology belongs to cytoplasmic engineering. The so-called nuclear transfer technology refers to the mechanical transfer of a nucleus (containing genetic material) called a "donor cell" to another cell called a "recipient" without a nucleus, and then the recombinant cell further develops and differentiates. The principle of nuclear transfer is based on the totipotency of animal nucleus.
The idea of cloning animals by nuclear transfer was first put forward by German embryologist in 1938. Starting from 1952, scientists first carried out nuclear transfer cloning experiments with amphibians, and successively obtained tadpoles and adult frogs. From 65438 to 0963, the research group led by Professor Tong Dizhou in China studied the nuclear transfer technology of fish embryos with goldfish as materials and achieved success. As of 1995, embryo nuclear transfer has been successful in major mammals, but nuclear transfer of differentiated cells in adults has not been successful.
1996, Roslin Institute, Edinburgh, UK, Ian? Wilmut's research team successfully bred Dolly the cloned sheep through nuclear transfer, which is the first cloned animal in the world to be nuclear transferred from adult mammalian somatic cells. .
Not all cells can be used as nuclear donors for nuclear transplantation. There are two kinds of donor cells: one is embryonic cells, and the other is somatic cells.
Studies have shown that eggs, oocytes and fertilized eggs are all suitable recipient cells.
In June, 2000, Northwest A&F University cloned two "cloned sheep" from adult goat somatic cells, which showed that scientists in China also mastered the cutting-edge technology of mammalian somatic cell nuclear transfer.
The study of nuclear transfer is not only of great scientific value to understand the totipotency of animal nucleus and the relationship between nucleus and cytoplasm, but also of great economic value and application prospect in animal husbandry production.
3. Cell fusion technology
Cell fusion technology belongs to cell fusion engineering. Cell fusion technology is a new technology to obtain hybrid cells and change their properties. It refers to the process of artificial fusion of somatic cells of the same or different species to form hybrid cells under in vitro conditions. Cell fusion is an important method in cytogenetics, cytoimmunology, virology and oncology.
The main steps of animal cell fusion are:
The first step is to obtain parental cells. Cells were isolated from the sampled tissues by trypsin or mechanical methods, and then cultured in monolayer culture or suspension respectively.
The second step is to induce fusion. The two parental cells were placed in the same culture medium for cell fusion. The fusion process of animal cells is generally as follows: two cells are in close contact → cell membrane is fused → channels or cell bridges appear between cells → the number of cell bridges increases to expand the channel area → two cells are fused into one.
The main steps of plant cell fusion are:
The first step is to prepare the parent protoplast.
The second step is to induce fusion.
The fusion steps of microbial cells are basically the same as those of plant cells.
Since 1970s, many kinds of cells have been successfully fused, including new hybrid plants such as "tomato and potato", "Arabidopsis rape" and "mushroom cabbage". (Figure 4-36 shows hybrid plants cultivated by cell fusion. ) At present, people can't fuse many distant cells into hybrid individuals, especially animal cells.
Enzyme engineering, fermentation engineering and protein engineering.
1, enzyme engineering Enzyme engineering refers to the use of specific catalytic functions such as enzymes, cells or organelles, and the use of biological reaction devices to produce products that human needs through certain technical means. It is a new technology formed by the combination of enzymology theory and chemical technology.
Enzyme engineering can be divided into two parts. Part is how to produce enzymes, and part is how to use enzymes.
The production of enzyme has roughly experienced four stages of development. Initially, enzymes were extracted from animal internal organs. With the development of enzyme engineering, people use a large number of cultured microorganisms to obtain enzymes. After the birth of genetic engineering, the enzyme-producing microorganisms were transformed by gene recombination. In recent years, a new hot topic has emerged in enzyme engineering, that is, artificial synthesis of new enzymes, that is, artificial enzymes.
The use of enzymes also has some disadvantages. If it encounters high temperature, strong acid and strong alkali, it will lose its activity, with high cost and price. In practical application, this enzyme can only be used once. Immobilization of enzyme can solve these problems, which is called the center of enzyme engineering.
In the early 1960s, scientists found that after immobilization, the activity of many enzymes did not decrease at all, but the stability was improved. This discovery is a turning point in the popularization and application of enzyme and the development of enzyme engineering. Nowadays, the immobilization technology of enzymes is changing with each passing day. It is manifested in two aspects:
One is the fixed method. At present, there are four fixing methods: adsorption method, valence bond method, crosslinking method and embedding method.
Secondly, there are many kinds of immobilized enzymes that can catalyze a series of reactions.
Compared with natural enzymes, immobilized enzymes and immobilized cells have obvious advantages:
1, which can be made into various shapes, such as granular, tubular, film, etc., and put into a reaction tank for easy taking out and continuous reuse;
2, the stability is improved, the activity is not easy to lose, and the service life is prolonged;
3. It is convenient for automatic operation and realizes computer-controlled continuous production.
At present, dozens of countries have used immobilized enzymes and immobilized cells in industrial production. The products include alcohol, beer, various amino acids, various organic acids and medicines.
2. Fermentation engineering
Modern fermentation engineering. Also known as microbial engineering, it refers to the use of modern bioengineering technology and some specific functions of microorganisms to produce products useful to human beings, or to directly apply microorganisms to industrial production processes.
Fermentation is a unique function of microorganisms, which has been recognized by human beings for thousands of years and used to make food such as wine and bread. In the 1920s, alcohol fermentation, glycerol fermentation and propanol fermentation were the main methods. The rise of American antibiotic industry in the mid-1940s, the large-scale production of penicillin and the success of glutamic acid fermentation in Japan greatly promoted the development of fermentation industry.
In 1970s, with the rapid development of biotechnology such as gene recombination and cell fusion, the fermentation industry entered the stage of modern fermentation engineering. It not only produces alcoholic beverages, acetic acid and bread, but also produces various medical and health care drugs such as insulin, interferon, growth hormone, antibiotics and vaccines, produces agricultural means of production such as natural pesticides, bacterial fertilizers and microbial herbicides, and produces amino acids, spices, biopolymers, enzymes, vitamins and single cell proteins in the chemical industry.
Broadly speaking, fermentation engineering consists of three parts: upstream engineering, fermentation engineering and downstream engineering. Upstream projects include the selection of excellent seed plants, the determination of optimal fermentation conditions (pH, temperature, dissolved oxygen, nutrients) and the preparation of nutrients. Fermentation engineering mainly refers to the technology of cultivating a large number of cells and producing metabolites in a fermentor under the best fermentation conditions. Downstream engineering refers to the technology of separating and purifying products from fermentation broth.
The steps of fermentation engineering generally include:
The first step is the breeding of strains.
The second step is the preparation and sterilization of the culture medium.
The third step is to expand culture and inoculation.
The fourth step is the fermentation process.
Step five, separation and purification.
Fermentation engineering has been widely used in many fields such as pharmaceutical industry, food industry, agriculture, metallurgical industry and environmental protection.
3. protein Project
In modern biotechnology, the protein project appeared in the early 1980s. Protein Project refers to the production of protein molecules with new structures and functions, which are not found in nature and useful for human life, on the basis of in-depth understanding of the spatial structure of protein and the relationship between structure and function, and mastering the technology of gene manipulation.
There are two main types of projects in protein:
One is to design from scratch, that is, to design and synthesize protein completely according to people's will. Ab initio design is the most important and difficult operation type in protein project. At present, the technology is not mature, and the synthesized protein is only a few short peptides.
The second is site-directed mutation and local modification, that is, only local modification is carried out on the basis of the existing protein. This technology, which aims to change the molecular structure of protein by inducing site-directed mutation of one or several bases, is called gene directed mutation technology.
The basic procedure of protein Plan is: firstly, determine the sequence of amino acids in protein, determine and predict the spatial structure of protein, establish the spatial structure model of protein, then put forward the idea of processing and transforming protein, obtain the required new protein gene through gene location mutation and other methods, and then synthesize protein. (Figure 4-37)
Because the protein Project was developed on the basis of genetic engineering, it has many similarities with genetic engineering technology in technology, so the protein Project is also called the second generation genetic engineering.
The protein Plan has found a new way to transform the structure and function of protein, which also indicates that it is possible for human beings to design and create excellent protein that does not exist in nature, thus generating huge social and economic benefits.