Method for constructing chicken-gene-EphA2-knocked-out cell line based on CRISPR-Cas9 editing technology

A construction method and cell line technology, applied in the field of cell line construction, to achieve the effect of large application research value

Inactive Publication Date: 2020-06-19
YANGZHOU UNIV
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  • Method for constructing chicken-gene-EphA2-knocked-out cell line based on CRISPR-Cas9 editing technology
  • Method for constructing chicken-gene-EphA2-knocked-out cell line based on CRISPR-Cas9 editing technology
  • Method for constructing chicken-gene-EphA2-knocked-out cell line based on CRISPR-Cas9 editing technology

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Embodiment Construction

[0028] The following examples further illustrate the technical solution of the present invention, but the content of the present invention is not limited thereto.

[0029] 1. Find the target gene: use the NCBI online database to search the chicken EphA2 genome sequence. NCBI ReferenceSequence: XM_001234813.5, design knockout target site in the third exon of the gene, the sequence of the third exon is shown in SEQ NO.1.

[0030] 2. Use the online design website to design sgRNA targeting the target sequence: use Zhang Feng’s laboratory website https: / / zlab.bio / guide-design-resources to design sgRNA, select the sgRNA with the highest score, and place it in front of the 5’ end of sgRNA_F CACCG was added at the end, AAAC was added to the 5' end of sgRNA_R, and C was added to the 3' end. The specific primer sequences are shown in Table 1, which were synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0031] SEQ ID NO.2 Table 1. sgRNA targeting chicken EphA2 gene

[0032] ...

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Abstract

The invention relates to a method for constructing a chicken-gene-EphA2-knocked-out cell line based on a CRISPR-Cas9 editing technology. The method comprises the principle and most-core key technologyas follows: the chicken-EphA2-knocked-out cell line is obtained through scientifically and reasonably constructing chicken-gene-EphA2-targeted sgRNA, then, transfecting sgRNA and Cas9 gene carried lentiCRISPR v2 plasmids, achieving gene silencing by using a CRISPR-Cas9 system, killing negative cells through drug screening, and then, acquiring monoclonal cell strains through a subcloning method. Through the invention, a method for targeted knockout of a chicken gene EphA2 based on the CRISPR-Cas9 and the sgRNA targeting to the chicken gene EphA2 are constructed, so that a chicken-gene-EphA2-knocked-out cell model is expectedly obtained and is applied to researches on related diseases. The construction of the chicken-gene-EphA2-targeted-knocked-out cell line based on the CRISPR-Cas9 is notreported in related fields, and the method will fill up vacancies of related technologies at home and abroad and has a relatively high application research value.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and relates to a method for constructing a cell line for knocking out chicken EphA2 gene based on CRISPR-Cas9 editing technology. Background technique [0002] The CRISPR-Cas system was first discovered in the natural immune system of bacteria, which is used to fight against virus invasion and foreign DNA. Among them, type II CRISPR-Cas system is the most widely used, mainly composed of RNA-guided Cas9 endonuclease, a guide RNA (sgRNA) and trans-activating RNA (tracrRNA). In this system, crRNA (CRISPR-derived RNA) combines with tracrRNA to form a double-stranded RNA through base pairing, guides the Cas9 protein to the target sequence, and specifically cuts the DNA double-strand at the 3bp upstream of the PAM sequence, resulting in a double-stranded DNA. Strand break (DSB, double strand break), thereby starting the DNA damage repair mechanism in the cell, resulting in base deletion or...

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Application Information

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IPC IPC(8): C12N5/10C12N15/90C12N15/85
CPCC12N5/0656C12N9/12C12N15/85C12N15/902C12N2510/00C12Y207/10001
Inventor 叶建强姚晓慧李拓凡万志敏邵红霞秦爱建
Owner YANGZHOU UNIV
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