Method and kit for marking glioma stem cell marker LGR5 by applying CRISPR/Cas9 and split GFP bimolecular fluorescence complementation technology

A glioma stem cell and bimolecular technology, applied in the field of methods and kits, can solve the problems of lack of highly specific antibodies, inability to accurately detect the expression level of LGR5 endogenous protein, etc., and achieve the effects of good stability and easy promotion.

Active Publication Date: 2022-04-15
SHAANXI NORMAL UNIV
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Problems solved by technology

[0005] The purpose of the present invention is to provide a method of applying CRISPR / Cas9 and split GFP bimolecular fluorescence complementary technology to label glioma stem cell marker LGR5 and its application, which integrates the latest endogenous protein labeling technology, namely CRISPR / Cas9 and split GFP Bimolecular fluorescence complementation technology, labeling endogenous LGR5 protein in glioma stem cells, overcomes the problem that existing methods cannot accurately detect the endogenous protein expression level of LGR5 in GSCs due to the lack of highly specific antibodies

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  • Method and kit for marking glioma stem cell marker LGR5 by applying CRISPR/Cas9 and split GFP bimolecular fluorescence complementation technology
  • Method and kit for marking glioma stem cell marker LGR5 by applying CRISPR/Cas9 and split GFP bimolecular fluorescence complementation technology
  • Method and kit for marking glioma stem cell marker LGR5 by applying CRISPR/Cas9 and split GFP bimolecular fluorescence complementation technology

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Embodiment 2

[0069] This example uses immunofluorescence and In situ analysis of the intracellular localization of LGR5 (labeled with GFP) and LPAR4 on frozen sections of GSCs and their induced glioma using PLA site ligation technique. Duolink The specificity and sensitivity of the site-connection technique are high. It uses a pair of DNA proximity probes, each consisting of a specific antibody (Ab-Oligo) conjugated to a high-affinity oligonucleotide. figure 2 Shown in A is the application Duolink The principle of detection of protein interaction by site-linking technology, the unmodified primary antibody needs to be recognized by the secondary antibody Ab-Oligo conjugate. When the distance between the two probes is very close, by adding DNA fragments that are complementary to the oligonucleotide sequence on the probe, the oligonucleotide on the probe will bind to the DNA through complementary pairing. Complementary, and then under the action of ligase, the oligonucleotides on the ...

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Abstract

The invention belongs to a glioma technology, particularly relates to a method and a kit for marking a glioma stem cell marker LGR5 by applying a CRISPR/Cas9 and split GFP bimolecular fluorescence complementary technology, and overcomes the problem that an existing method cannot accurately detect the endogenous protein expression level of the LGR5 in GSCs due to lack of a high-specificity antibody. According to the invention, the CRISPR/Cas9 technology is ingeniously applied to mark the LGR5 protein with a smaller subunit GFP11, and GSCs for stably expressing the larger subunit GFP1-10 protein are constructed at the same time. When the two fragments are in the same cell, the two parts can stably interact to generate GFP with a luminescence function, so that the endogenous expression level of LGR5 in glioma stem cells can be detected. The detection method is good in stability and convenient to popularize.

Description

technical field [0001] The invention belongs to glioma technology, and in particular relates to a method and a kit for labeling glioma stem cell marker LGR5 using CRISPR / Cas9 and split GFP bimolecular fluorescence complementary technology. Background technique [0002] Glioblastoma (Glioblastoma multiforme, GBM) is the most common and most malignant brain tumor of the adult central nervous system. Due to the highly invasive growth of malignant glioblastoma, the boundary between the tumor and normal brain tissue is unclear, making it difficult to completely remove it by surgery. Although the clinical treatment techniques based on radiotherapy and chemotherapy have been improved and improved, the recurrence of glioblastoma patients is still inevitable, the median survival period is only 15 months, and the five-year survival rate after diagnosis is less than 5%. . Therefore, for highly heterogeneous glioblastoma, it is of great research significance to discover effective ther...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486Y02A50/30
Inventor 解源张磊张研宇贺琪媛张凌雪王思男
Owner SHAANXI NORMAL UNIV
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