Method for rapid identification of proliferation phenotype in cell line after esophagus cancer functional gene knockout

Abstract

The invention belongs to the technical field of molecular biology and relates to a method for rapid identification of a proliferation phenotype in a cell line after esophagus cancer functional gene knockout. By a stream type cell sorting instrument, 200 positive cell populations with carrier fluorescent tags are sorted into two holes of a 96-well plate, then FA (fragment analysis) is performed after 0h culture, and then effective editing clone of a target locus can be detected after CRISPR-Cas9 targeting of the functional gene; after culturing for 7 days, fragment analysis is performed again,a fragment analysis peak can be varied according to different cell proliferation characteristics, cells high in proliferation speed are remarkable in peaking while cell populations low in proliferation speed are not obvious in peaking or even have no peak, and thus, the proliferation phenotype after esophagus cancer functional gene knockout can be quickly judged.

Description

technical field [0001] The invention relates to a rapid identification method for the proliferation phenotype after the functional gene knockout of esophageal cancer in a cell line, and belongs to the technical field of molecular biology. Background technique [0002] Esophageal cancer is one of the most common malignant tumors in my country. Because of its indistinct early symptoms, most patients with esophageal cancer are diagnosed at an advanced stage, and the prognosis is extremely poor. At present, the 5-year survival rate of patients with esophageal cancer in my country is only about 20%. Despite the grim situation, in the field of tumor research, the basic research and drug development of esophageal cancer are relatively lagging behind, especially the distribution of esophageal cancer in the world has significant differences, and China is the main high-incidence area. Most are squamous cell carcinomas. Therefore, it is necessary to develop a high-throughput research...

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Problems solved by technology

[0004] For traditional cell line knockout, even if the CRISPR / Cas gene editing system is used, it is still necessary to perform multiple rounds of resistance screening after cell transfection and limit dilution to isolate monoclonal cells and expand the culture before performing phenotypic analysis. The dilution method is still a resistance screening method. After screening positive cells, it is necessary to expand and culture a single cell to a certain extent before performing phenotype analysis, which consumes a lot of time and labor costs; moreover, the screening efficiency is low, and the analysis of phenotype is time-consuming and expensive. To a certain extent, the progress and breadth of research on functional genes of esophageal cancer have been delayed

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