Method for constructing 14-3-3 epsilon gene knockout cell strain based on CRSIPR technology and application of 14-3-3 epsilon gene knockout cell strain

A gene knockout, cell line technology, applied in applications, animal cells, genetic engineering, etc., can solve the problems of HP-PRRSV replication increase, and achieve the effect of short cycle, low cost and stable cell line

Active Publication Date: 2021-07-30
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Using the 14-3-3 inhibitor Difopein TFA or siRNA to interfere with 14-3-3ε can inhibit the replication of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), and overexpression of 14-3-3ε leads to HP- Increased PRRSV replication

Method used

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  • Method for constructing 14-3-3 epsilon gene knockout cell strain based on CRSIPR technology and application of 14-3-3 epsilon gene knockout cell strain
  • Method for constructing 14-3-3 epsilon gene knockout cell strain based on CRSIPR technology and application of 14-3-3 epsilon gene knockout cell strain
  • Method for constructing 14-3-3 epsilon gene knockout cell strain based on CRSIPR technology and application of 14-3-3 epsilon gene knockout cell strain

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Embodiment 1

[0028] Example 1 Construction of 14-3-3ε gene knockout cell line based on CRSIPR technology

[0029] 1. sgRNA design and synthesis

[0030] Download the corresponding nucleotide sequence according to the chicken source 14-3-3ε (ENSGALG00000002661) gene published by ensembl, and use the online sgRNA design tool (http: / / crispor.tefor.net) in the second exon region of 14-3-3ε ) designed 14-3-3ε sgRNA (small guide RNA), and the primers were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis. The synthetic primer sequences are as follows:

[0031] sgRNA-F: 5'-caccgGGTTGAATCAATGAAGAAAG-3';

[0032] sgRNA-R: 5'-aaacCTTTCTTCATTGATTCAACCc-3'.

[0033] 2. Construction of recombinant eukaryotic expression plasmid pX459-14-3-3ε

[0034] Annealing: After centrifuging the sgRNA sequence synthesized by the company, add an appropriate amount of ddH 2 O, make the final concentration 100 μmol / L, add T4 ligase after fully dissolving, and use PCR instrument to anneal the sgRNA ...

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Abstract

The invention discloses a method for constructing a 14-3-3 epsilon gene knockout cell strain based on a CRSIPR technology and application of the 14-3-3 epsilon gene knockout cell strain, and belongs to the technical field of biology. The invention provides an sgRNA sequence with 14-3-3 epsilon gene knocked out. The nucleotide sequence of the sgRNA sequence is as shown in SEQ ID NO: 1. The 14-3-3epsilon gene is knocked out on a chicken fibroblast line DF-1 cell genome for the first time by utilizing a CRISPR-Cas9 technology, so that the expression of 14-3-3epsilon protein is completely lost, and the 14-3-3epsilon knocked-out DF-1 cell strain is obtained; wherein the activity, the growth speed and the like of the knockout cell strain are not obviously different from those of a control cell, and the DF-1 knockout cell model is an ideal DF-1 knockout cell model. The method is simple and convenient to operate, short in period, low in cost and stable in cell strain after modification, and can be used for researching the biological function of the 14-3-3 epsilon protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing a 14-3-3ε gene knockout cell line based on CRSIPR technology and an application thereof. Background technique [0002] 14-3-3 proteins are highly conserved in most animals and plants, and there are seven subtypes of 14-3-3 proteins (β, γ, ε, σ, ζ, τ, and η) in mammalian cells. Each isoform of the 14-3-3 family can form a homo- or hetero-dimer, and as a molecular chaperone protein, it mainly binds to phosphorylated proteins and regulates the cellular sublocalization of its target proteins after being stimulated, thereby regulating such as interferon A variety of important cellular physiological processes such as cell reaction and apoptosis. Studies have shown that the Zika virus NS3 protein antagonizes retinoic acid-inducible gene-1 (RIG-1) and melanoma differentiation-associated molecules (MDA) by binding and sequestering 14-3-3ε and 14-3-3η pro...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N15/12C12N5/10
CPCC12N15/113C12N15/85C07K14/47C12N5/0656C12N2310/10C12N2510/00Y02A50/30
Inventor 任涛张殿宸梁健鹏向斌林秋燕
Owner SOUTH CHINA AGRI UNIV
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