Method for constructing stable-expression SpCas9 protein cell line and application

A construction method and cell line technology, which are applied in the field of cell line construction with stable expression of SpCas9 protein

Inactive Publication Date: 2021-05-18
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] In the existing sgRNA verification and functional gene screening technology systems, most of them use a single-plasmid system to express SpCas9 protein. Generally, the single-plasmid system is a eukaryotic expression system, which results in some primary cells or some cell lines with low transfection efficiency. Effective sgRNA functional verification and functional gene screening cannot be performed

Method used

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  • Method for constructing stable-expression SpCas9 protein cell line and application
  • Method for constructing stable-expression SpCas9 protein cell line and application
  • Method for constructing stable-expression SpCas9 protein cell line and application

Examples

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Embodiment 1

[0047] This example is the construction of a mouse myoblast C2C12 cell line stably expressing SpCas9 protein. The cell line is mainly constructed by lentiviral infection. The lentiviral expression plasmid used is LentiCas9-Blast, and Addgene official website number is #52962.

[0048] First, package and purify the lentivirus:

[0049] (1) HEK 293T cell preparation: Because the lentiviral packaging process is highly dependent on cells, it is necessary to select HEK 293T cells with a clear source and good growth status, and use the mycoplasma inhibitor Biomyc-3 (purchased from Biological Industries company); the method of use is to add it to the culture medium at a ratio of 1:100 during the cell culture process. Two passages were processed to ensure cell status.

[0050] (2) Transfection of packaging plasmids: Transfection was carried out according to the cation-mediated cell transfection method, specifically ① inoculate HEK 293T cells of appropriate density before transfection...

Embodiment 2

[0066] This example is the construction of a cell line stably expressing SpCas9 protein from E14 embryonic stem cells. The cell line is mainly constructed by lentivirus infection. The lentivirus expression plasmid used is LentiCas9-Blast, and Addgene official website number is #52962.

[0067] First, package and purify the lentivirus:

[0068] (1) HEK 293T cell preparation: Because the lentiviral packaging process is highly dependent on cells, it is necessary to select HEK 293T cells with a clear source and good growth status, and use the mycoplasma inhibitor Biomyc-3, a product of Biological Industries, to resuscitate the recovered cells ; The method of use is to add it to the culture medium at a ratio of 1:100 during cell culture. Two passages were processed to ensure cell status.

[0069] (2) Transfection packaging plasmid: Transfection is carried out according to the cation-mediated cell transfection method. The ratio of the viral packaging plasmid is LentiCas9:psPAX2:pMD...

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Abstract

The invention relates to a method for constructing a stable-expression SpCas9 protein cell line and application. The construction method comprises the following steps of: packaging and purifying lentivirus, namely preparing HEK 293T cells, transfecting packaging plasmids, performing transfection culture, collecting cell culture supernatant, and concentrating virus liquid; and infecting cells with the obtained concentrated virus, namely screening drug concentration, infecting cells with lentivirus, screening drugs, and selecting monoclone for identification. By adopting the method for constructing the stable-expression SpCas9 protein cell line, a mouse myoblast C2C12 stable-expression SpCas9 protein cell line and a mouse embryonic stem cell E14 stable-expression SpCas9 protein cell line are obtained, SpCas9 protein can be stably expressed, and the cell line can serve as a rapid verification tool, is used for performing rapid functional verification on designed SgRNA so as to detect whether SgRNA design is successful, and can serve as a functional gene screening system for subsequent large-scale functional gene screening.

Description

technical field [0001] The invention relates to a method and application for constructing a cell line stably expressing SpCas9 protein, and belongs to the field of cell biology technology. Background technique [0002] In the existing sgRNA verification and functional gene screening technology systems, most of them use a single-plasmid system to express SpCas9 protein. Generally, the single-plasmid system is a eukaryotic expression system, which results in some primary cells or some cell lines with low transfection efficiency. Effective sgRNA functional verification and functional gene screening cannot be performed. How to effectively express the SpCas9 protein is an urgent technical problem to be solved. Contents of the invention [0003] (1) Technical problems to be solved [0004] In order to solve the above problems in the prior art, the present invention provides a method for constructing a cell line stably expressing SpCas9 protein, and provides mouse myoblast C2C1...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867C12N5/077C12N5/0735
CPCC12N9/22C12N5/0658C12N5/0606C12N15/86C12N2740/10043C12N2510/00
Inventor 张业孙文政代辉
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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