echemi logo
Product
  • Product
  • Supplier
  • Inquiry
    Home > Biochemistry News > Biotechnology News > The basic procedure of gene therapy.

    The basic procedure of gene therapy.

    • Last Update: 2020-10-20
    • Source: Internet
    • Author: User
    Search more information of high quality chemicals, good prices and reliable suppliers, visit www.echemi.com

    "name

    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:

    rgb (70,135

    ,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

    < "middle" > use
    the name Mechanism transfeding efficiency
    < td align " "middle" > advantages and disadvantages< td align " "middle" > factors calcium phosphate transfeding method internal swallowing effect 20% Cell transflyction scient expression1, simple and effective

    2, suitable for walling, non-walled cells

    3, often preferred method

    1, Ca2 plus, DNA concentration

    2, PH value, precipitation reaction time

    3, chloroquine, glyceloride and sodium butyrate treatment

    DEAE glucosamine transfed Inhibition of nuclease

    insitionation

    higher in BDC-1, CV-1 and COS cells instantaneous expression simple operation1, high concentration of DNa

    2, DEAE glucosaccharide concentration

    3, temperature and education time

    Poly-brene transfeding method is not clear. Low molecular weight DNA transfeding rate is higher than calcium phosphate method stabilize converters of CHO cells can get more stable converters 1, subject cell type

    2, transfeding regulatory signal

    3, DNA concentration

    protosite fusion cell membrane fusion 50-100% transflysing, stable rate of 00%. .2-0.02% speed expression

    stable expression

    1, operation complex

    2, can not be transfected

    3, carefully used to screen nutritional defect variants

    1, lysosomes, PEG concentration

    2, temperature and culture time

    electrient perforation high pressure on the membrane to form micro-perforation. high efficiency instantaneous expression

    steady transformation

    1, precision instruments

    2, can be used in animals, plant cells and bacteria

    1, electric field strength

    2, pulse length

    3, temperature, DNA concentration

    4, culture fluid

    lipid body lipid membrane fusion 50-60% transfected instantaneous expression

    stabilized transformation

    1, simple operation

    2, can be used for in vivo testing

    1, liposome mass

    2, DNA concentration

    cytocytocytotylete microinfring method direct injection50-100% transfed . Stable conversion 1, the need for special instruments

    2, the number of processing samples is less

    3, efficient

    1, injection technology

    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.

    This article is an English version of an article which is originally in the Chinese language on echemi.com and is provided for information purposes only. This website makes no representation or warranty of any kind, either expressed or implied, as to the accuracy, completeness ownership or reliability of the article or any translations thereof. If you have any concerns or complaints relating to the article, please send an email, providing a detailed description of the concern or complaint, to service@echemi.com. A staff member will contact you within 5 working days. Once verified, infringing content will be removed immediately.

    Contact Us

    The source of this page with content of products and services is from Internet, which doesn't represent ECHEMI's opinion. If you have any queries, please write to service@echemi.com. It will be replied within 5 days.

    Moreover, if you find any instances of plagiarism from the page, please send email to service@echemi.com with relevant evidence.