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Method for targeting knockout of chicken IRF7 gene and application thereof in vaccine preparation

A gene and gene knockout technology, applied in the field of genetic engineering, can solve the problems of low knockout efficiency and achieve high efficiency and fast speed

Active Publication Date: 2019-07-26
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] CRISPR / Cas9 has been used for gene knockout in various model animals, however, the application of this technology in avian gene knockout is still seldom, and the existing applications in poultry have reported low knockout efficiency

Method used

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  • Method for targeting knockout of chicken IRF7 gene and application thereof in vaccine preparation
  • Method for targeting knockout of chicken IRF7 gene and application thereof in vaccine preparation
  • Method for targeting knockout of chicken IRF7 gene and application thereof in vaccine preparation

Examples

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Embodiment 1

[0061] Example 1, the method of using CRISPR / Cas9 technology to knock out the IRF7 gene of DF-1 cells

[0062] 1. Design of gRNA targeting chicken IRF7

[0063] Search the gene sequence of chicken IRF7 through GeneBank, and its sequence is shown in SEQ ID NO.1.

[0064] Exons of this gene were used as target design regions. Schematic diagram of the gRNA sequence designed for the chicken IRF7 gene based on CRISPR / Cas9 figure 1 As shown, the designed gRNA sequence is:

[0065] IRF7-gRNA1:AAGGCCAGCGGCAGGTACGAGGG SEQ ID NO.2;

[0066] IRF7-gRNA2: AAGGGATGCGGAAGATACGGCGG SEQ ID NO.3.

[0067] 2. Construction of CRISPR / Cas9 targeting vector

[0068] According to the gRNA1 and gRNA2 sequences designed in 1, respectively design and synthesize two pairs of reverse complementary oligonucleotides, the synthesized two pairs of oligonucleotide sequences are:

[0069] 1) 5'AAGGCCAGCGGCAGGTACGAGGG 3'SEQ ID NO.4;

[0070] 3'TTCCGGTCGCCGTCCATGCTCCC 5' SEQ ID NO.5;

[0071] 2) 5'AAGGG...

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Abstract

The invention discloses a method for targeting knockout of a chicken IRF7 gene and application thereof in vaccine preparation. Based on CRISPR / Cas9, the gRNA target sequence targeting the chicken IRF7gene is designed and ligated to a VK001-02 plasmid to obtain a chicken IRF7 gene targeting vector; the targeting vector is transfected into chicken fibroblasts using liposomes. After successfully transfected cells are screened by puromycin, the chicken fibroblasts with the chIRF7 gene knocked out are screened by a limited doubling dilution method. A knockout cell line is verified by TCID50 to indicate the increase in virus titer. The method is applicable to the study of the role and the function of the important transcription factor IRF7 in the interferon expression pathway mediated by a chicken natural immune pattern recognition receptor. At the same time, the knockout cell line constructed can down regulate the expression level of the interferon beta mediated by many congenital immunitypathways, which is beneficial to virus propagation and can be used for virus amplification in vaccine preparation.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for targeted knockout of chicken IRF7 gene using CRISPR / Cas9 technology and its application in vaccine preparation. Background technique [0002] CRISPR / Cas9 is an innate immune system widely found in bacteria and archaea. The basic structure of the system includes tracrRNA sequence region, cas gene sequence region and CRISPR sequence region. When the phage invades the host for the first time, the protein complex encoded by the cas gene lock cleaves the terminal spacer sequence on the phage DNA, and then integrates the cleaved fragment into the CRISPR 5' end of its own DNA. When the phage invades the host again, the CRISPR sequence region transcribes pre-crRNA, which matures into crRNA under the joint action of RNase III, cas9 and tracrRNA. The crRNA and tracrRNA form a complex, which is complementary to the specific site of the phage DNA and bi...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/90C12N5/10C12Q1/686
CPCC07K14/4702C12N15/113C12N15/85C12N15/907C12N2310/10C12Q1/686C12N2310/20C12Q2531/113
Inventor 孙建和程玉强伦敏翔严亚贤
Owner SHANGHAI JIAO TONG UNIV
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