Method for performing high-throughput screening on gene deletion mutation

A screening method, gene deletion technology, applied in the direction of mutant preparation, etc., can solve the problems of high antibody preparation cost, large amount of antibody complement, long cycle, etc., to achieve the effect of improving accuracy and sensitivity, avoiding complement toxicity, and high sensitivity

Inactive Publication Date: 2017-06-13
HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0010] Although this experimental system is relatively mature, and its detection sensitivity is much higher than that of the HGPRT experiment and the bacterial Ames experiment, there are still some disadvantages, such as the cumbersome operation of the early mutation experiment and the need to consider the lethality of complement itself on cells; It takes 10-12 days for cell cloning culture, which is a long cycle, and the cell phenotypes of different mutation degrees are consistent, and they all form similar clonal groups and are not easy to distinguish; this method is limited by the area of ​​the culture dish and the number of planted cells, and only a limited number of s

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  • Method for performing high-throughput screening on gene deletion mutation
  • Method for performing high-throughput screening on gene deletion mutation
  • Method for performing high-throughput screening on gene deletion mutation

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Embodiment 1

[0053] A high-throughput screening method for gene deletion mutations in this embodiment is applicable to the mutagenicity analysis of various environmental pollutants such as asbestos, organic pollutants, heavy metals, radiation, etc., and includes the following steps:

[0054] (1) Cell treatment and continuous culture

[0055] Normal culture two dishes A L cells (including wild-type A L cells from wild-type A L Cell development of various defective cells and constructed cell lines) to the logarithmic growth phase were divided into control group and treatment group, that is, one dish was not treated, and the other dish was continuously treated with 2 μg / mL sodium arsenite for 24 hours (induced mutation), then control group and treatment group A L Each cell is 2.0×10 5 / ml cell density was replanted into the culture dish and cultured continuously for 7 days, so that the surviving cells after treatment had enough time to recover from the temporary stagnation of growth, and ...

Embodiment 2

[0078] A high-throughput screening method for gene deletion mutations in this embodiment is applicable to the mutagenicity analysis of various environmental pollutants such as asbestos, organic pollutants, heavy metals, radiation, etc., and includes the following steps:

[0079] (1) Cell treatment and continuous culture

[0080] Normal culture two dishes A L cells (including wild-type A L cells from wild-type A L Cell development of various defective cells and constructed cell lines) to the logarithmic growth phase were divided into control group and treatment group, that is, one dish was not treated, and the other dish was continuously treated with 1 μg / mL sodium arsenite for 48 hours (induced mutation), then control group and treatment group A L Each cell is 3.0×10 5 / ml cell density was replanted into the culture dish and cultured continuously for 6 days, so that the surviving cells after treatment had enough time to recover from the temporary stagnation of growth, and ...

Embodiment 3

[0100] A high-throughput screening method for gene deletion mutations in this embodiment is applicable to the mutagenicity analysis of various environmental pollutants such as asbestos, organic pollutants, heavy metals, radiation, etc., and includes the following steps:

[0101] (1) Cell treatment and continuous culture

[0102] Normal culture two dishes A L cells (including wild-type A L cells from wild-type A LCell development of various defective cells and constructed cell lines) to the logarithmic growth phase were divided into control group and treatment group, that is, one dish was not treated, and the other dish was continuously treated with 4 μg / mL sodium arsenite for 12 hours. (induced mutation), then control group and treatment group A L Each cell is divided into 5.0×10 5 / ml cell density was replanted into the culture dish and cultured continuously for 8 days, so that the surviving cells after treatment had enough time to recover from the temporary stagnation of...

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Abstract

The invention discloses a method for performing high-throughput screening on gene deletion mutation. The method comprises the following steps: performing cell treatment and continuous cultivation, collecting cells, binding with fluorescent antibodies, preparing upper sample suspension, and performing cell sorting, cell proliferation and atlas analysis. The method has the following advantages: (1) the method is simple in operation, the whole process of collecting the cells, binding with specific fluorescent protein and sorting only needs 3 to 4 hours, and the detection and screening efficiency of the CD59 gene deletion mutation is improved; and (2) CD59-phenotype single cells with different mutation types and capable of being continuously cultivated can be obtained quickly, the experiment sensitivity and accuracy of a mutation analysis system are effectively improved, and high-throughput screening characteristic is embodied.

Description

technical field [0001] The invention relates to the technical field of mammalian cell mutation detection and screening, in particular to a high-throughput screening method for gene deletion mutations. Background technique [0002] At present, cancer has been recognized as the number one killer that seriously endangers human health in the world. Environmental pollutants are necessary for the occurrence of cancer by inducing gene mutations and causing irreversible damage to genetic material. Therefore, the objective evaluation of the genotoxicity of environmental pollutants, especially their mutagenicity, has important guiding significance for the early warning of human cancer. In recent years, A L Cells have been widely used to study the mutagenicity and related mechanisms of environmental pollutants such as asbestos, organic pollutants, heavy metals, radiation, and nanomaterials. A L Cells are Chinese hamster CHO cells gly - The immortalized cells obtained by hybridizin...

Claims

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Application Information

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IPC IPC(8): C12N15/01
CPCC12N15/01
Inventor 许安王希楠陈少鹏
Owner HEFEI INSTITUTES OF PHYSICAL SCIENCE - CHINESE ACAD OF SCI
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