Experimental method for constructing Hutat2:Fc gene knock-in monocyte by using CRISPR/Cas9 technology

A monocyte and gene knock-in technology, applied in the medical field, can solve the problems of difficult operation of gene-directed insertion technology, unable to express genes stably for a long time, affecting the stability of the genome, etc., to protect the migration ability, wide application range, increase effect of possibility

Active Publication Date: 2018-09-21
FOURTH MILITARY MEDICAL UNIVERSITY
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Problems solved by technology

Virus-mediated transfection technology has obvious biosafety problems, and the random integration of genes caused by it may affect the stability of the genome; liposome-mediated cell transfection technology cannot express therapeutic genes stably for a long time; ZFN / TALEN-mediated Guided gene-directed insertion technology is difficult to operate and the editing efficiency is low

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  • Experimental method for constructing Hutat2:Fc gene knock-in monocyte by using CRISPR/Cas9 technology
  • Experimental method for constructing Hutat2:Fc gene knock-in monocyte by using CRISPR/Cas9 technology
  • Experimental method for constructing Hutat2:Fc gene knock-in monocyte by using CRISPR/Cas9 technology

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Embodiment Construction

[0042]CRISPR / Cas9 technology is a newly discovered gene editing technology in recent years, which can realize gene editing at specific sites in the genome. Compared with ZFN / TALEN-mediated gene-directed insertion technology, CRISPR / Cas9 technology is easy to operate and has high editing efficiency. It can realize site-directed single-copy gene insertion and has little impact on genome stability. Moreover, the inherent AAVS1 site in the human genome sequence is a "safe harbor" site in the human genome sequence, which can realize the insertion of various foreign genes without affecting the normal function of cells. Therefore, the present invention uses this technology to insert the therapeutic gene into the AAVS1 site of human primary mononuclear cells, so that it can pass through the blood-brain barrier and enter the nervous system to exert a therapeutic effect.

[0043] Utilize CRISPR / Cas9 technology to construct Hutat2: the experimental method of Fc gene knocking into monocyt...

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Abstract

The invention relates to an experimental method for constructing a Hutat2:Fc gene knock-in monocyte by using a CRISPR / Cas9 technology. The experimental method is characterized by comprising the following steps of (1) CRISPR / Cas9 targeting plasmid construction; (2) donor plasmid construction; (3) primary generation monocyte sorting; (4) monocyte transfection through electroporation; and (5) knock-in efficiency identification. The experimental method for constructing the Hutat2:Fc gene knock-in monocyte by using the CRISPR / Cas9 technology has the advantages that the method is simple and implemented easily, subsequent cell screening is not needed, the higher gene editing efficiency can be obtained, the feasibility and application prospect of the experiment are greatly increased, and the application range is wide.

Description

technical field [0001] The present invention relates to an experimental method, in particular to an experimental method for constructing Hutat2:Fc gene knock-in monocytes using CRISPR / Cas9 technology. Belongs to the field of medicine. Background technique [0002] In recent years, the infection rate of acquired immunodeficiency virus (HIV) has increased year by year. With the discovery and popularization of antiviral drugs, HIV infection has gradually changed from an acute fatal disease to a chronic and controllable disease. With the prolongation of patients' survival time, the incidence of complications of HIV infection has increased year by year, among which HIV-related neurocognitive disorders have received widespread attention. The incidence of HIV-related neurocognitive disorders in HIV-infected patients is 40%-60%, which greatly affects the quality of life of patients. [0003] Studies have found that the HIV transcriptional activator (HIV-Tat) plays an important ro...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/90
CPCC12N5/0645C12N15/907C12N2510/00C12N2800/80C12N2810/10
Inventor 康文王博文孙永涛左佳蕙康文臻
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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