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gene therapy is developed on the basis of genetic engineering "> molecular biology technology, it is more complex than other existing treatments. The basic process of gene therapy includes the following main aspects:
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,221); For genetic diseases, wild-type genes can be used for gene therapy, such as ADA gene therapy for ADA defects, as long as it has been studied that the occurrence of a disease is caused by a genetic abnormality. But under the present conditions, this alone is not enough.
gene that can be used for gene therapy needs to meet the following points:
in the body only a small amount of expression can significantly improve symptoms; It is < certain genes such as > , such as insulin genes associated with blood sugar concentration, are not currently available for gene therapy for diabetes.
In gene therapy of antivirals and pathogens, the chosen target gene should play an important role in the life history and function of the virus and pathogen and the sequence is specific, such as the HBeAg or X gene for HBV.
Tumor patients have many immunodeficiency, optional immune factor gene into the human body, tumor cells often exist in a variety of abnormal forms of genes, can use antisanthorization technology to close the cancer gene in the cell or into the cell into the wild type anti-cancer gene, inhibit tumor growth, the target cancer gene or anti-cancer gene should be under the occurrence and development of the tumor has a clear correlation.
the gene to be selected, the gene of the purpose is prepared. The genes that are expressed positively can be c DNA, or genomic DNA fragments. It can be obtained by traditional methods, or it can be amplified in-body using new technologies such as polymerase chain reaction ". Some anbolic genes can also be obtained by this method, but in most cases by synthetic preparation.
(ii) genes
there are now a variety of gene transport methods, the basic principle is to transport the foreign gene into the cell. There are two major categories of virus vectors and non-viral vectors used.
virus vectors: viruses have some unique properties such as most viruses can infect specific cells, not easily degraded within the cells, RNA viruses can be integrated into chromosomes and high gene levels. Therefore, viral vector is a good gene transport vector. Viruses currently used as vectors include retrovirus, adenovirus, and gland-related viruses. Herpes virus and hepatitis virus, etc. Retrovirus is used as a vector in several steps:
(1) transforms natural wild preRNA viruses into DNA vectors and inserts the relevant foreign genes to be transferred. The basic principle is to replace the encoded genes of the virus with labeled genes and extrogengenes.
(2) prepares auxiliary cells to provide the lost function of vector DNA.
(3) the carrier DNA is imported into the auxiliary to produce the virus vector.
(4) virus vector infects target cells, and exgengencies are expressed within the cells.
non-viral vectors: the rapid development of such vectors, currently mainly liposomes, some vectors with the function of the carrier also presents a seductive prospect, about the advantages and disadvantages of common vectors listed in Table 2.
23-2 Advantages and disadvantages of common gene transport vectors
"firstRow | disadvantages | ||
reverse pathogenic toxicity | The genome is small and simple stable integration into the host genome biological characteristics are clear cells that can be efficiently transferred to replication nocuous to host cells | infection only to split cells random integration (which can lead to mutations) often only short-lived expressions and low viral titularity (107pfu/ml) . Virus recombinant insertion capacity may be limited (10kb) | |
drogen-related viruses | genome small (5kb) seedable integration into human chromosome 19 human cells as host notyviral, non-pathogenic | has not been studied clearly densomal-assisted replication carrying external gene capacity limited (3) 4kb) It is difficult to obtain high-titration viruses | |
addrogen viruses | suitable for in-place use, especially lung (highly efficient in vivo infection in non-dividing cells) virus titration high (1010pfu/ml) biological characteristics are clear | not integrated with the host genome (only short-term expression) loaded genome complex. virus proteins may cause immune and inflammatory responses limited ability to insert exogenetic genes (7-8kb) | |
liposomes | inoculative capacity theoretically no DNA size limit low toxicity | not specific target cells low trans-infection efficiency only short-lived expression difficult to apply in vivo | |
receptively mediated transport | no infection ability specific trans-infective target cells theoretically no DNA size limit build flexibility | low trans-contamination efficiency in vivo application difficulty there may be immunogenicity only short-term expression |
(iii) target cell selection
Theoretically, no matter what cell has the ability to accept external DNA, currently Gene therapy prohibits the use of reproductive cells as target cells, but only the use of somogenetic cells, for genetically modified so many cells must be easy to obtain, rich in content, easyculture, long life. The selected cells are lymphocytes, hematocytes, endocal cells, horn cells, endostrotes, fibroblasts, liver cells, muscle cells, and tumor cells. In practice, the application should be selected according to the conditions of the purpose.
(iv) cell transfection
there are many ways to import the target gene into the target cell, which can be broadly divided into four categories: physical method, chemical method, fusion method and viral infection method. The virus method is described in the "Transfer of Genes" in this section. At present, more use is the lipid body method.
23-3 DNA is a common method for importing mammalian cells
the name | Mechanism | transfeding efficiency | < "middle" > use
2, suitable for walling, non-walled cells
3, often preferred method
2, PH value, precipitation reaction time
3, chloroquine, glyceloride and sodium butyrate treatment
insitionation
2, DEAE glucosaccharide concentration
3, temperature and education time
2, transfeding regulatory signal
3, DNA concentration
stable expression
2, can not be transfected
3, carefully used to screen nutritional defect variants
2, temperature and culture time
steady transformation
2, can be used in animals, plant cells and bacteria
2, pulse length
3, temperature, DNA concentration
4, culture fluid
stabilized transformation
2, can be used for in vivo testing
2, DNA concentration
2, the number of processing samples is less
3, efficient
2, DNA concentration
in the current state of technology, in general, its gene trans-trans-transtation efficiency is difficult to reach 100%. Therefore, it is necessary to distinguish between transducting cells and unconducted cells first. New technologies in this area have developed rapidly, and commonly used transduction cell screening methods are:
using gene expression product screening:
(1) marker gene screening: the introduction of a marker gene in the vector, or at the same time the introduction of marker genes, the appropriate time after transfilction to choose the appropriate dose< a href""> medium, screening for labeled gene esotypes, those cells that have been imported into the exgengengenic gene will survive, while unretred cells will die from selectivecell culture base. If neor marker genes are present in more vectors, if G418 is added to the medium for selection, only transduction cells survive.
(2) Selective of gene defective-type subject cells: the selection of gene-defective cells is used after the normal gene is imported into the gene-defective target cells, and selective media are used for screening. For example, the TK gene is imported into the target cells of TK-, transcription cells can grow in the HAT medium, and unconducted cells cannot grow in the HAT medium.
(3) Gene co-transfect technology: the target gene expression vector DNA and marker gene expression vector DNA mixed together transferred to the target cell, respectively, using the marker gene and the target gene corresponding to the selector for two screening, and finally obtained a compound transduction of the converter.
molecular biology method: whether the exogengenated gene is really transferred to the target cell must be confirmed by molecular hybridization. Common methods are in-place hybridization, Southern hybridization and dot hybridization. The main problem is the choice of probe. If the transferred target gene does not exist in the target cell, the target gene can be used.