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A method for simultaneously knocking out EGFRWT and EGFRVIII in glioblastoma

A technology for glioblastoma and glioma cells, applied in the field of DNA recombination, can solve the problem of inability to block or inhibit EGFRwt at the same time, and achieve the effect of inhibiting the occurrence and development of tumors

Active Publication Date: 2019-12-31
GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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Problems solved by technology

[0005] In order to solve the problem that the existing technical means cannot simultaneously block or inhibit the expression of EGFRwt and EGFRvIII, the present invention proposes a method for simultaneously knocking out EGFRwt and EGFRvIII in glioblastoma

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  • A method for simultaneously knocking out EGFRWT and EGFRVIII in glioblastoma
  • A method for simultaneously knocking out EGFRWT and EGFRVIII in glioblastoma
  • A method for simultaneously knocking out EGFRWT and EGFRVIII in glioblastoma

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Embodiment Construction

[0020] The present invention will be further described below with reference to the drawings and embodiments.

[0021] 1. Cas9 and sgRNA lentivirus design and packaging

[0022] Cas9 plasmid was purchased from addgene, and EGFR sgRNA was designed from http: / / crispr.mit.edu / based on the EGFR sequence (NM_005228), and the highest-scoring sequence was taken and located on chromosome 17: AGATCCCGTCCATCGCCACT. The sgRNA sequence was ligated using GV371 plasmid.

[0023] Cell preparation:

[0024] 1) Discard the old culture medium, add 5 ml of sterilized PBS solution, shake gently to wash the cell growth surface, and then discard the PBS solution.

[0025] 2) Digest 293T cells (ATCC) in logarithmic growth phase with 2 ml trypsin (Gibco, Thermo Fisher Scientific, USA).

[0026] 3) Adjust the cell density to 5×10 with a medium containing 10% serum 6 Cells / 10ml, re-seeded in 100 mm cell culture dish, 37℃, 5% CO 2 Continue to culture, the cell density before transfection is about 80%.

[0027] Pla...

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Abstract

The invention discloses a method for knocking out EGFRwt and EGFRvIII simultaneously from glioblastoma multiforme and relates to the recombinant DNA technology. The target site of sgRNA in an EGFR gene is on exon 17 of the EGFR gene, and is a gene sequence expressing a transmembrane region of the EGFR. According to the method for constructing Cas9 and sgRNA lentiviruses and infecting the glioblastoma multiforme with the lentiviruses, EGFRwt and EGFRvIII can be knocked out simultaneously from the glioblastoma multiforme. EGFRwt and EGFRvIII can be knocked out simultaneously and stably from the glioblastoma multiforme with the method, and occurrence and development of tumor can be inhibited effectively after knockout. The sgRNA is expected to be applied to novel drugs for treating EGFR amplification type and EGFRvIII mutant type glioblastoma multiforme.

Description

Technical field [0001] The present invention relates to DNA recombination technology, more specifically a method for simultaneously knocking out EGFRwt and EGFRvIII in glioblastoma. Background technique [0002] Gliomas are the most common primary tumors in the human skull, and glioblastomas are the most malignant. Despite the use of advanced surgical treatment, radiotherapy and chemotherapy, the average survival period of glioblastomas remains at Around 14 months, the treatment of glioblastoma is a world problem. Of patients with glioblastoma, 40-60% have EGFR amplification, and about 25% have EGFR type III mutations (EGFRvIII). Both conditions enhance EGFR activity, enhance tumor malignancy and reduce patients The prognosis. At present, EGFR monoclonal antibodies (mAb) and small molecule inhibitors (TKI) for EGFR amplification and mutant treatment have been successful in lung cancer, but because of the existence of the blood-brain barrier, mAbs and TKIs cannot reach glioblast...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/867C12N5/10
CPCC12N15/1138C12N15/86C12N2310/10C12N2740/15043C12N2800/107C12N2800/80
Inventor 康春生黄凯王蕴非王琦雪杨超
Owner GENERAL HOSPITAL OF TIANJIN MEDICAL UNIV
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