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Porcine fibroblast line with double sgRNA, gene knockout vector and gene knockout STING gene and construction method of porcine fibroblast line

A gene knockout and carrier technology, applied in the field of porcine fibroblast cell lines and its construction, can solve problems such as the inability to guarantee the automatic repair of cells, the inability of cell lines to be cloned, and the risk of uncertainty in cloning operations

Active Publication Date: 2021-08-20
ANIMAL SCI RES INST GUANGDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0010] 1. The cell lines prepared by the above method cannot be cloned
[0011] 2. The method of using the above-mentioned lentiviral vector will introduce foreign genes, and there is a great risk of uncertainty in the subsequent cloning operation
[0012] 3. The above method only knocks out the Sting gene, and cannot guarantee that the cells will not automatically repair the deletion in the subsequent cloning process

Method used

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  • Porcine fibroblast line with double sgRNA, gene knockout vector and gene knockout STING gene and construction method of porcine fibroblast line
  • Porcine fibroblast line with double sgRNA, gene knockout vector and gene knockout STING gene and construction method of porcine fibroblast line
  • Porcine fibroblast line with double sgRNA, gene knockout vector and gene knockout STING gene and construction method of porcine fibroblast line

Examples

Experimental program
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Effect test

Embodiment 1

[0057] Construction of CRISPR / Cas9 targeting vector targeting STING gene

[0058] 1. Use the NCBI database to search the gene sequence of porcine STING (Gene ID: 100217389), and locate the first exon (fourth exon) and the last exon ( Exon 8) segment for target design.

[0059] The sgRNA was designed using the website http: / / crispor.tefor.net / . The sgRNA fragments targeting the fourth exon were named: STING-sgRNA1, STING-sgRNA2, STING-sgRNA3; the sgRNA fragments targeting the eighth exon were named: STING-sgRNA4, STING-sgRNA5. Add cohesive ends to both ends of the sgRNA according to the BbsI restriction endonuclease. The method is: add the base CACCG at the 5' end of the sgRNA sequence to obtain a forward fragment; if the first base at the 5' end of the sgRNA sequence is not G, then Add a CAAA base sequence to the 3' end of the sgRNA reverse complementary strand and add a C base to the 5' end as a reverse fragment; if the first base at the 5' end of the sgRNA sequence is G, t...

Embodiment 2

[0101] Construction of porcine fibroblast cell line with gene knockout of STING gene

[0102] (1) The above plasmids with high cutting efficiency (the expression vector group screened in Example 1, PX459-STING-sgRNA2+PX459-STING-sgRNA4 group). The cells were re-transfected by liposome transfection, and 24 h after transfection, the transfected cells were treated with 1.5 μg / mL puromycin for 3 days. After two rounds of selection, all the cells in the control group died. After the cells in the experimental group were digested, monoclonal cells were picked by the limiting dilution method and expanded for culture.

[0103] (2) Identify all the mononuclear clonal cells picked, and extract the genomic DNA of the cells for PCR identification. The primers used for PCR identification were STING-F1: 5'-TGGATTTCTTGGTTCCTACAG-3' (nucleotide sequence No. 11) and STING-R1: 5'-CGAGTCCTCACCCTGGTAGA-3' (nucleotide sequence No. 12).

[0104] The amplification conditions were 95°C, 5min; 95°C, ...

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Abstract

The invention belongs to the technical field of biology, and discloses double sgRNA which is a combination of one of STNG-sgRNA 1-3 targeting a fourth exon and one of STNG-sgRNA 4-5 targeting an eighth exon. After the double sgRNA is carried on an expression vector, an appropriate gene knockout vector can be obtained, the STING gene of the porcine fibroblast is knocked out in a large fragment mode, and therefore the obtained porcine fibroblast line has the advantages of being suitable for cloning, free of knockout gene repairing capacity and free of exogenous gene introduction. Meanwhile, the invention also discloses a construction method of the porcine fibroblast line.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a double sgRNA, a gene knockout vector, a pig fibroblast cell line with a gene knockout STING gene and a construction method thereof. Background technique [0002] my country is the largest pork production and consumption country in the world. With the improvement of people's living standards, our demand for high-yield and high-quality pigs is gradually increasing. As a key link connecting the lives of Chinese residents and the healthy development of the pig industry, pig products are the focus of my country's animal husbandry industry for their quality and safety. The occurrence of common pig diseases such as swine fever, suis streptococcal disease, and piglet diarrhea has seriously affected my country's pig industry and seriously endangered the safety of my country's pig industry. The No. 194 Announcement of the Ministry of Agriculture and Rural Affairs of my country has clearly p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/12C12N5/10C12R1/91
CPCC07K14/70539C12N5/0656C12N15/1138C12N15/85C12N2510/00C12N2800/107C12N2310/20
Inventor 王塑天张昆丽杨烨城黄秋艳孟繁明李剑豪
Owner ANIMAL SCI RES INST GUANGDONG ACADEMY OF AGRI SCI
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