In 1907, the American scholar R. G. Harrison first put nerve cell It survived in vitro. In 1956, American scholar T. T. Parker made single mammalian somatic cells divide and proliferate successfully under the conditions of in vitro culture microbiology Methods The materials for somatic cytogenetic research under strictly controlled conditions simplified the acquisition of higher animals in vitro Somatic cloning The research of somatic cytogenetics has been pushed to a new stage. In the early 1960s, French scholar G. Basqui and others found that mouse cells cultured in vitro could spontaneously fuse. Later, Japanese scholar Yoshio Okada found that the cells inactivated by ultraviolet light Sendai virus Can promote cell fusion 。 In the following years, three major advances have been made in the research of somatic cytogenetics: ① the introduction of the Recessive mutation cell line As the parent of cell fusion; When two different recessive mutant cells are mixed, only the fused cells can Mutant The functional complementation of cells wild type Characteristics, and then these fusion cells can be selected from a large number of unfused cell populations by various selection techniques; ② After obtaining different species nucleus Interspecific difference of Nuclear cell Later, we obtained the Synucleation Cells, that is, the nuclei of different species fuse into one nucleus Hybrid cell ；③ Found two species of cell fusion Later, hybrid cells always continuously reject chromosomes of one species during division. For example, mouse cells and Human cells The hybrid cells formed tend to constantly reject human chromosomes during division. On the basis of the above three findings, the human Gene mapping Working, normal somatic cells and tumor cell Study on tumorigenicity of hybrid cells and nuclear cytoplasm The study of the relationship with certain chromosomes. In 1975, Argentine scholar C. Milstein and others produced a large number of anomalies immunoglobulin Mice of Myeloma cells Mice immunized with specific antigens spleen lymphocyte conduct Somatic hybridization , obtained from hybrid myeloma cells cell line , greatly improving the purity of the antibody immunology and Medical Science Has had a significant impact. In addition, various methods are used to transfer genetic material between somatic cells in order to observe the effect of foreign genes host cell The fate of. In 1967, voles were first transformed by microorganisms Melanoma Cellular Deoxyribonucleic acid (short for DNA ）Transforming non Melanocyte To make it possible to produce melanin And proliferated into clones. In the 1970s, some new technologies were developed, such as Microcell 、 Liposome and Blood shadow cell And other carriers, such as several chromosomes, chromosome fragments or DNA molecule introduce Receptor cell ； Or by Microinjection hold DNA molecule Inject directly into the nucleus of the receptor cell, and finally make these introduced exogenous genetic materials express in the receptor cell, thus greatly promoting the Gene structure And features and gene regulation Research on. The research work of plant somatic cytogenetics is plant tissue culture Developed on the basis of. In 1934, British scholar P · R · White built the first cell that can actively grow with tomato root as material Asexual line 。 The future development is mainly about the tissue differentiation and cell fusion Two aspects. In 1956, R. A. Miller discovered Kinetin , and when containing a certain concentration of kinetin and auxin Organ differentiation of tissue cultured in vitro can be achieved on the medium of. Cells from tissue cultures of more than 200 species and genera have been induced to differentiate into plants. In the early 1960s, E · C · Kejin and others applied cellulase Detached plant Protoplast get ahead. In 1972, through selective screening, S · Carlson obtained Hybrid cell It is a diploid tobacco plant. Since the 1970s cell line Screening, introduction of exogenous genetic material and advanced plant cell The research on plasmids has also been widely carried out as well as animal somatic cytogenetics. Especially because plant cells have Omnipotence It can grow from a single cell into a plant, so plant somatic cytogenetics research is not only to clarify the organ Differentiation and Morphogenesis It is also of great significance to breeding work. 
Somatic cytogenetics use Inactivation Virus (such as Sendai virus ）Or Chemicals (e.g polyethylene glycol 、 glucan ）And other treatment cells can promote the fusion between cells to obtain different cell hybrids. For example, it contains more than two same species nucleus Homotypic nuclear cells of Synucleation Cell Cytoplast Fused with intact cells Cytoplasmic hybrid Cells and cytoplasm of one cell without cytoplasm Of Nucleosome Recombinant cells fused. These cell hybrids obtained through asexual process can be used to study the relationship between nucleus and cytoplasm genetic information It can also be used in plants to overcome Distant hybridization Difficulties. mutagenesis
use Induced mutation The mutation mechanism can be studied and cell fusion provide Selective marker 。 Established mutation cell line The traits of Radiation resistance , antiviral and drug resistance, etc. In plants, somatic mutation can also be used for breeding. Selection method of fusion cells
Cell fusion and Gene mutation It is also a rare event, so it is necessary to have a specific selection method to separate fused cells from a large number of non fused cells. stay Animal cell The earliest and most widely used HAT selection method 。 In this method, one parent cell line is Hypoxanthine - Guanine -Phosphoribosyltransferase deficiency（ HGPRT -）； The other parent cell line is Thymidine Kinase deficiency (TK -), when containing hypoxanthine (H) Aminopterin （A）、 thymus In HAT selective culture medium of azodinucleoside (T), none of the above parent cells can survive, only the fused Hybrid cell So hybrid cells can be effectively selected. The principle of HAT selection method is that nucleic acid There are two ways of synthesis: ① total synthesis, first synthesis from some small molecules purine 、 pyrimidine , and finally synthesize nucleic acid; ② Emergency route, through HGPRT (hypoxanthine guanine phosphate Ribose Converting enzyme ）The catalytic effect of Hypoxanthine Convert to Hypoxanthine nucleoside -Phosphoric acid (IMP) via TK（ Thymidine Kinase) to transform thymidine into Deoxythymidine -Phosphoric acid (dTMP), and then further synthesize nucleic acid. HAT culture medium Aminopterin (A) can block the total synthesis pathway, so neither parent cell can HAT medium Medium growth, but only by cell fusion The hybrid cells obtained after HGPRT And TK enzymatic activity Only then can hypoxanthine (H) and thymidine (T) in the culture medium be used to synthesize through emergency pathway nucleic acid (See figure). Similarly, any other method can be used to select fusion cells as long as it has the condition that the two parent cells can not survive alone but can only survive after fusion. Methods of excluding or retaining a parent chromosome
For the long term Subculture Mice and Rat cell line In terms of fusion cells, it is rat chromosomes that are constantly rejected in the process of passage; In the fusion cells of mouse and human, human chromosomes are rejected. However, if cells from human cell lines are fused with mouse cells that have not been subcultured for a long time, the fused cells will reject mouse chromosomes first. In plants, soybean chromosomes were retained in soybean and tobacco hybrid cell lines after nine months of culture, while tobacco chromosomes were excluded in half. According to the characteristics of some genes on the chromosome, we can selectively exclude Hybrid cell , for example, in the first Chromosome 5 There is one on Diphtheria toxin Receptor protein gene, which makes human hybrid cells sensitive to diphtheria toxin. Mouse cells do not have this gene, so they can resist this toxin. Fusion of human and mouse cells cell culture In the culture medium containing diphtheria toxin, hybrid cells with human chromosome 5 can be selectively removed. On the contrary, fusion cells can selectively retain a certain chromosome. for example HGPRT Gene located in human X chromosome Yes, so I can HAT culture medium The fusion cells growing in the cell must retain the human X chromosome. Chromosome gene or nucleus Transfer of Introducing genetic material into the cells of a certain organism is a common means in somatic cytogenetics research. Direct transformation of exogenous genetic material Receptor cell Efficiency is not high. Use virus Blood shadow cell 、 Microcell 、 Liposome After loading, it can improve the efficiency of introducing receptor cells. In addition, very thin glass tubes can also be used to inject DNA directly into the nucleus of receptor cells. After removing the cell wall plant cell Of Protoplast It can also be introduced by the above methods DNA molecule 、 phage Granules and organelles. In addition to transferring chromosomes or chromosome fragments, sometimes it is necessary to transfer the whole nucleus ， Microinjection It is also a common method of transferring nucleus. application cell fusion 、 Chromosome identification , biochemical identification, immunological identification and other technologies have been established Gene mapping Methods, the research of human gene mapping has made rapid progress. For example, Chinese hamster cells can be used to fuse with human somatic cells Hybrid cell stay Subculture In the process, the phenomenon of constantly rejecting human chromosomes is used to carry out gene positioning: for example, human chromosomes in hybrid cells are found Chromosome 9 Lost after being rejected ABO blood group antigen The ABO blood group antigen gene is equal to chromosome 9. Research tumor cell The change of tumorigenicity of hybrid cells formed by fusion can help us understand normal cells The canceration and reversal of tumor cells provide important clues. Somatic cytogenetic methods can also be applied to tumors (e.g Xeroderma pigmentosum ）Study on the mechanism of occurrence. In addition, the method of somatic cell culture can also be applied to Human genetic diseases Prevention of. Introduction of genetic material Receptor cell After that, analyze whether foreign genes are expressed in receptor cells and understand gene expression The necessary condition is to study Eukaryotic cell gene regulation Effective way. In addition, research nucleus and cytoplasm The relationship between them and the effect of cytoplasm on nuclear gene expression are also important contents of somatic cytogenetics research. The application of somatic cytogenetics in breeding is limited to plants. In the cell line of the medicinal plant Coptis chinensis, the total alkaloid content was up to 15% of the dry weight (berberine content was up to 8%) cell line These cell lines can be used for berberine fermentation. The cell lines resistant to Helminthosporium longum were also selected from the somatic cells of maize through mutagenesis and bred into resistant strains. adopt Protoplast fusion It can also obtain Distant hybridization Strains such as potato and tomato, tobacco and Petunia hybrida A hybrid line of. With the deepening of research work, more achievements will be achieved in production practice. 
