Establishment method of positive screening system based on gene knockout cells

Abstract

The invention belongs to the technical field of zoobiology, and relates to an establishment method of a positive screening system based on gene knockout cells; on the basis of the established system,p53 and MAD2 genes are knocked out in NIH / 3T3 cells, and the cells, which undergo gene knockout, are subjected to positive screening. According to the establishment method, target sequences of the sixth exon of the p53 gene and the first exon of the MAD2 gene are subjected to site-directed knockout in an NIH / 3T3 gene line, and in the combination with a green fluorescent protein gene and a puromycin gene, the cells, which undergo p53 and MAD2 gene knockout, are subjected to positive screening; and by virtue of the established positive screening system, the p53 and MAD2 genes are simultaneouslyedited in living mouse liver, so that a mouse liver cancer model is finally obtained. The method provided by the invention lays a foundation for related researches on editing the p53 and MAD2 genes ina living mouse.

Description

Technical field [0001] The invention belongs to the field of animal biotechnology, and relates to a method for establishing a positive screening system based on gene knockout cells, and specifically relates to a knockout of p53 and MAD2 genes in NIH / 3T3 cells and positive screening of cells with gene knockout Method of system establishment. This method uses the established system to knock out the p53 and MAD2 genes in NIH / 3T3 cells, and positively screen the cells with gene knockout. Background technique [0002] The prior art discloses that liver cancer is one of the cancers with a high mortality rate in the world. Among men and women, liver cancer is the third and sixth highest mortality cancer, which has seriously threatened human health and life. According to statistics, the 5-year average survival rate of liver cancer patients is less than 11%; about 700,000 new liver cancer patients and nearly 600,000 patients die of liver cancer worldwide each year; my country is the coun...

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Problems solved by technology

[0004] Studies have disclosed that MAD2 (mitotic arrest defect protein 2) is an important part of the mitotic checkpoint complex protein, which is located on human chromosome 4; the tumor suppressor gene p53 is involved in regulating cell cycle progression, DNA repair and apoptosis, and Cell transformation is closely related to the occurrence of tumors. Wild-type p53 ensures normal cell cycle progression by regulating the G1-S checkpoint and maintaining G2-M arrest, thereby maintaining normal cell growth, chromosome stability or under various cellular stresses. Induced cell apoptosis; In human liver cancer tissues, the loss of MAD2 and tumor suppressor gene p53 is common; the abnormal expression of MAD2 is closely related to cancer, and some cells lacking MAD2 (MAD2 + / - ) is accompanied by the occurrence of chromosomal instability, which eventually leads to the formation of lung cancer in mice, and the overexpression of MAD2 in mice can also lead to chromosomal instability, aneuploidy of cells and the occurrence of tumors; a large number of studies have shown that chromosome Instability is one of the main driving forces leading to tumorigenesis. In addition, it has been confirmed in mouse embryonic fibroblasts that the simultaneous knockout of MAD2 and the tumor suppressor gene p53 can lead to extreme chromosomal instability.

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