Method for establishing HK2 report gene cell line of colon cancer

Abstract

The invention discloses a method for establishing an HK2 report gene cell line of the colon cancer. The method comprises the specific steps of firstly designing a specificity sgRNA sequence of a HK2 gene site, and performing cloning into a plasmid PX459; integrating a homologous recombination sequence of an HK2 gene and a green fluorescent protein DNA fragment (EGFP), and performing joint electricshock on the plasmid and the integrated fragment to transform a colon cancer cell line HCT116; performing unicellular screening on EGFP expression cells through a flow cytometer, and amplifying a monoclonal cell line; and performing PCR identification and immunoblotting verification on the screened EGFP expression cell line so as to obtain a positive HK2 report gene cell line. The colon cancer cell line cell line HK2 gene and EGFP are in co-expression, and the EGFP expression level and the HK2 gene have high consistency, so that through detecting EGFP expression level changes, the HK2 gene expression level can be accurately judged. The method for establishing the cell line is simple, and easy to operate, and gene sites can be efficiently and accurately positioned.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a colorectal cancer HK2 reporter gene cell line and its establishment method. Background technique [0002] The energy required for cell survival mainly comes from glycolysis in the cytoplasm and aerobic oxidation in the mitochondria. In the 1920s, German biologist Otto Warburg discovered that tumor cells would use glucose for glycolysis and produce lactic acid under aerobic conditions. A new way of thinking. The research results of Gillies RJ et al. show that in normal tissues, 10% of the total ATP synthesized by cells is provided by the glycolytic pathway, and mitochondria provide the remaining 90% of the energy, while in tumor cells the energy provided by the glycolytic pathway accounts for 50% -70%, the additional energy comes from mitochondria (Cancer and Metastasis Reviews, 2007.26(2):311-317.2). Existing studies have proved that hexokinase is the first rate-limiting key enz...

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Problems solved by technology

[0005] However, there is currently no rapid and efficient method for establishing a stable cell line of the colorectal cancer HK2 reporter gene. This study focuses on the field of the colorectal cancer HK2 reporter gene, using CRISPR-Cas9 technology to construct a cell line with enhanced green fluorescent protein (EGFP ) cell line of the colorectal cancer HK2 reporter gene, which provides a favorable tool for the study of the HK2 gene and its signaling pathway, the study of the pathogenic mechanism of related diseases, drug screening and evaluation

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