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Double sgRNA, gene knockout carrier, gene knockout sting gene pig fibroblast cell line and construction method thereof

A gene knockout, porcine fibroblast technology, applied in the field of porcine fibroblast cell line and its construction, can solve the problems such as the inability to guarantee the automatic repair of cells, the inability to clone the cell line, and the uncertainty risk of the cloning operation.

Active Publication Date: 2021-10-15
ANIMAL SCI RES INST GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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  • Double sgRNA, gene knockout carrier, gene knockout sting gene pig fibroblast cell line and construction method thereof
  • Double sgRNA, gene knockout carrier, gene knockout sting gene pig fibroblast cell line and construction method thereof
  • Double sgRNA, gene knockout carrier, gene knockout sting gene pig fibroblast cell line and construction method thereof

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Experimental program
Comparison scheme
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 field of biotechnology, which discloses a double sgRNA, which is one of the STING-sgRNA 1-3 targeting the fourth exon and one of the STING-sgRNA 4-5 targeting the eighth exon combination of one. After the double sgRNA is carried on the expression vector, a suitable gene knockout vector can be obtained to knock out the STING gene of porcine fibroblasts with a large fragment, and the resulting porcine fibroblast cell line is suitable for cloning and has no knockout gene repair ability 1. A porcine fibroblast cell line without exogenous gene introduction. At the same time, the invention also discloses a method for constructing the porcine fibroblast cell 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 Patents(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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