A method for establishing hk2 reporter gene cell line of colorectal cancer

A reporter gene and cell line technology, applied in the field of colorectal cancer HK2 reporter gene cell line and its establishment, can solve the problem of not establishing a colorectal cancer HK2 reporter gene, and achieve the promotion of drug metabolism evaluation and related gene function research, time-consuming Few, high success rate effect

Active Publication Date: 2019-10-08
GUANGDONG MEDICAL UNIV
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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

Method used

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  • A method for establishing hk2 reporter gene cell line of colorectal cancer
  • A method for establishing hk2 reporter gene cell line of colorectal cancer
  • A method for establishing hk2 reporter gene cell line of colorectal cancer

Examples

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Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Establishment of colorectal cancer HK2 reporter gene cell line

[0044] Step 1: Design and evaluate suitable HK2-sgRNA sequences:

[0045] Through screening and evaluation, two sgRNA sequences were obtained, which are:

[0046] HK2-sgRNA1: TAGAACCCCTGAAATCGGAA (chr2:74890939-74890958), as shown in SEQ ID NO.1.

[0047] HK2-sgRNA2: TGTGTCAGAGACAGACCCCT (chr2: 74891015-74891034), as shown in SEQ ID NO.2.

[0048] Step 2: Construct pX459-sgRNA plasmid, including the following steps:

[0049] According to the sequence of HK2-sgRNA1 or HK2-sgRNA2, the plasmid pX459 / HK2-sgRNA1 or pX459 / HK2-sgRNA2 with the correct sgRNA sequence can be directly obtained after synthesis by the biological company.

[0050] Step 3: Integrate the L-EGFP-R fragment, including the following steps:

[0051] According to the homology arm of the HK2 gene and the EGFP sequence, the correct integrated fragment L-EGFP-R was directly obtained after being synthesized by the biological company....

Embodiment 2

[0064] Example 2 Functional verification of colorectal cancer HK2 reporter gene cell line

[0065] Design 3 different small interfering RNAs specifically targeting the HK2 gene, such as Image 6 The shown shRAN-1, shRAN-2, and shRAN-3 were respectively transfected into the HK2 reporter cell line. After 72 hours, the analysis of the transcription level showed that compared with the non-knockdown control, the three specific small interfering RNAs effectively reduced the expression level of the HK2 gene (approximately 70-80% knockdown); at the same time, EGFP gene expression is also correspondingly reduced by 70-80% along with HK2 gene knockdown. The experimental results prove from the molecular level that the reporter gene EGFP and the HK2 gene in the colorectal cancer HK2 reporter gene cell line constructed by the present invention can be synchronously co-expressed, and can be synchronously inhibited by shRNA, which can be used for tracking the suppression or overexpression of ...

Embodiment 3

[0066] Example 3 drug screening and evaluation verification

[0067] 2-Methoxyestradiol (2-MeOE2) is a HIF inhibitor, which has entered the phase II anti-tumor clinical trial. Using this inhibitor to treat the HK2 cell line, it was found that the expression of EGFP and HK2 were jointly inhibited, see Figure 7 . After the HK2 cell line was treated with 2-MeOE2 (10uM) for 24h and 48h, the relative expression changes of HK2 and EGFP transcript levels compared with the untreated control were analyzed. The results showed that the expression level of HK2 was significantly inhibited, and the expression of EGFP was also synergistically inhibited; The experimental results further prove that the HK2 reporter gene cell line of colorectal cancer constructed by the present invention can be used to screen and evaluate related anticancer drugs.

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Abstract

The invention discloses a method for establishing a HK2 reporter gene cell line of colorectal cancer, specifically: first designing the HK2 gene site-specific sgRNA sequence, and cloning it into the plasmid PX459; integrating the HK2 gene homologous recombination sequence and the green fluorescent protein DNA fragment ( EGFP), the above-mentioned plasmids and integrated fragments were electroporated together to transform the colorectal cancer cell line HCT116; the cells expressing EGFP were screened by flow cytometry, and the monoclonal cell lines were amplified; the screened EGFP expressing cell lines were identified by PCR and immune Blot validation of positive HK2 reporter gene cell lines. The colorectal cancer cell line co-expresses HK2 gene and EGFP, and its EGFP expression level is highly consistent with HK2 gene, so the expression level of HK2 gene can be accurately judged by detecting the change of EGFP expression level. The method for establishing a cell line of the present invention is simple, easy to implement, highly efficient and accurately locates a gene locus.

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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/90C12Q1/02C12R1/91
CPCC12N2503/02C12N2510/00C12N5/0693C12N15/907C12N2310/20C12Q1/6897
Inventor 余红兵刘欣
Owner GUANGDONG MEDICAL UNIV
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