Method for performing high-throughput screening on gene deletion mutation

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...

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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 single cells can be obtained. Mutant cell clone population; the process of picking single-cell clones can easily cause contamination between different single-clones, which requires very high technical requirements for experimental operators. In order to ensure that the analyzed cells come from independent single-cell clones, each culture dish Only one or two single-cell clones are isolated in the experiment, which greatly increases the workload; the rabbit serum complement in the traditional experiment is expensive, the cost of antibody preparation is high, and the amount of antibody complement is large, and the equipment such as cell culture dishes is more expensive Wait

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