Cell line for knocking out chicken Shp-2 gene based on CRISPR-Cas9 editing technique and construction method of cell line

A technology of shp-2 and construction method, applied in the field of genetic engineering

Inactive Publication Date: 2020-01-14
YANGZHOU UNIV
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Problems solved by technology

Numerous studies have shown that the expression of Shp-2 and the change of its activity are related to malignant proliferation of hematopoietic cells, human Noonan syndrome, childhood leukemia, various human malignancies, autoimmune diseases and invasion of some viral infections. There are few reports on the function research of source Shp-2

Method used

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  • Cell line for knocking out chicken Shp-2 gene based on CRISPR-Cas9 editing technique and construction method of cell line
  • Cell line for knocking out chicken Shp-2 gene based on CRISPR-Cas9 editing technique and construction method of cell line
  • Cell line for knocking out chicken Shp-2 gene based on CRISPR-Cas9 editing technique and construction method of cell line

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Embodiment Construction

[0028] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0029] 1. Find the target gene: use the ensembl online database to search the chicken Shp-2 genome sequence ENSGALG00000004821.5, Transcript ID: ENSGALT00000007704.4, design the knockout target site in the second exon of the gene, and the first exon The sub sequence is shown in SEQ ID NO.1.

[0030]2. Design of sgRNA and synthesis of oligonucleotide chain: use the website (http: / / crispr-era.stanford.edu / index.jsp and http: / / crispr.mit.edu) to analyze and obtain the specific sgRNA score , select the 5 sgRNAs (sgRNA-1, sgRNA-2, sgRNA-3, sgRNA-4, sgRNA-5) in the second exon with the highest score as alternative sequences. Add CACC to the 5' end of the sgRNA sense strand template; add C to the 5' end of the antisense strand template, and add AAAC to the 3' end, which match the sticky end after lentiCRISPRv2 digestion. A total of 5 primers were designed, and Suzhou Jin ...

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Abstract

The invention relates to a cell line for knocking out a chicken Shp-2 gene based on a CRISPR-Cas9 editing technique and a construction method of the cell line. The cell line adopts the principle and the core key technology that the sgRNA of a target chicken Shp-2 gene is scientifically and reasonably constructed, then the sgRNA is cloned to a lentiCRISPRv2 plasmid with a Cas9 gene and the plasmidis transfected into cells. A CRISPR-Cas9 system is used for gene silencing, and a drug screening and subcloning method is adopted, so that finally, a positive monoclonal cell strain is obtained, and achicken source Shp-2 knockout cell line is obtained. The invention aims to construct a chicken Shp-2 gene target knockout method based on CRISPR-Cas9, and sgRNA of the target chicken Shp-2 gene, so as to obtain a chicken source Shp-2 gene knockout cell model, and to be used for research of relevant diseases. The construction of the cell line for knocking out chicken Shp-2 gene based on a CRISPR-Cas9 is not reported in related fields. The cell line can fill the blank of correlation techniques at home and abroad, and has great application and research value.

Description

technical field [0001] The invention relates to a cell line for knocking out chicken Shp-2 gene based on CRISPR-Cas9 editing technology and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] The CRISPR-Cas system is an acquired immune system that widely exists in bacteria and archaea, and plays an important role in bacteria's resistance to foreign pathogens and plasmid transformation. At present, it has been found that there are 6 types of CRISPR-Cas, Ⅰ-Ⅵ, and CRISPR / Cas9 is transformed from the type Ⅱ system. The CRISPR / Cas9 system consists of the Cas9 protein with nuclease activity, the crRNA transcribed from CRISPR, and the tracrRNA complementary to the transactivation CRISPR repeat region. Among them, crRNA combines with tracrRNA to form double-stranded RNA through base pairing, recruits Cas9 protein to the target gene site to exert endonuclease activity, and specifically cuts the target double-stranded ...

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Application Information

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IPC IPC(8): C12N5/10C12N15/113C12N15/85C12N15/90
CPCC12N5/0693C12N9/16C12N15/113C12N15/85C12N15/907C12N2310/20C12N2510/00C12Y301/03048
Inventor 叶建强谢菁谢泉邵红霞秦爱建万志敏
Owner YANGZHOU UNIV
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