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M6A'reader 'YTHDF2 gene modification method and application thereof

A genetic modification and reader technology, applied in the field of genetic engineering, can solve problems such as unclearness and virus influence, achieve wide application prospects, and change the effect of virus multiplication ability

Pending Publication Date: 2021-06-11
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been found that there are multiple m6A modification sites on many viral RNAs (Lichinchi G,, et al.Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells.Naturemicrobiology.2016; 1(4 ):16011), but it is still unclear how m6A modification affects viral replication

Method used

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  • M6A'reader 'YTHDF2 gene modification method and application thereof
  • M6A'reader 'YTHDF2 gene modification method and application thereof
  • M6A'reader 'YTHDF2 gene modification method and application thereof

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Embodiment example 1

[0036] Implementation case 1: CRISPR / Cas9 transformation of Vero81 cell line YTHDF2 protein

[0037] The cell genetic modification method of the embodiment of the present invention:

[0038] 1. According to the coding region and non-coding region of the YTHDF2 gene sequence of the African green monkey (Chlorocebus sabaeus), after a comprehensive comparison, select the third coding region and the front 1 / 3 region of the fourth coding region for sgRNA sequence design, and design according to the sgRNA In principle, three sgRNAs were selected for efficiency verification. The sgRNA selection principle in this embodiment is that the GC content is moderate (40%-60%), and the selected region is located at the front end of the gene.

[0039] In this implementation case, the sgRNA sequence of YTHDF2 is as follows:

[0040] sgRNA1-F (SEQ ID NO.3): 5'-CACCGTCTTATGGACAACTGAGCAA-3' and sgRNA1-R (SEQ ID NO.4): 5'-CTTGCTCAGTTGTCCATAAGACAAA-3';

[0041] sgRNA2-F (SEQ ID NO.5): 5'-CACCGAGAC...

Embodiment example 2

[0051] Implementation Case 2: Detection of YTHDF2 Protein Expression in Transformed Vero81 Cells

[0052] 1. Verification of pCas9-YTHDF2 plasmid cutting efficiency.

[0053] Take out the Vero81 cells from the liquid nitrogen tank and quickly place them in a flowing water bath at 37°C. After about 2-3 minutes to melt completely, wipe the outside of the cryopreservation tube with alcohol cotton on the ultra-clean bench of the cell room, and transfer it to a 25mL cell culture bottle for 4 hours. After the cells adhered to the wall, the corresponding fresh complete medium was replaced, and after the cells were overgrown, the cells were subcultured at a ratio of 1:3. 24 hours before the experiment, the Vero81 cells were inoculated into a 6-well cell culture plate and cultured until the cell density reached 70%-80% confluence. Take out the pCas9-YTHDF2-1, pCas9-YTHDF2-2, pCas9-YTHDF2-3 and pCDNA3.1-mcherry-YTHDF2 plasmids, and use NanoDrop2000 to measure the nucleic acid concentra...

Embodiment 3

[0058] Embodiment 3: the influence of the transformed Vero cell YTHDF2 protein on virus proliferation

[0059] Knockout of YTHDF2 gene can specifically affect the proliferation of porcine epidemic diarrhea virus (PEDV). Real-time fluorescent quantitative PCR amplification was used to compare the expression of YTHDF2 gene in wild-type Vero81 cells and the expression of porcine epidemic diarrhea virus N gene in transformed Vero81 cells. from Figure 7 It can be seen that compared with wild-type Vero81 cells, the proliferation effect of porcine epidemic diarrhea virus in the transformed Vero81-YTHDF2-KD cells is significantly higher than that in wild-type cells. This shows that knocking out YTHDF2 can significantly promote virus replication.

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Abstract

The invention provides an m6A'reader 'YTHDF2 gene modification method and application thereof, and relates to the technical field of gene engineering. According to the gene modification method, a gene editing technology is used for modifying a YTHDF2 gene to obtain a cell line with the YTHDF2 gene knocked out, and the gene modification method comprises the steps of sgRNA sequence design, YTHDF2 gene knockout, plasmid construction and modified gene recombination cell line screening. According to the invention, the cells with specific genes are subjected to gene modification, the fact that the YTHDF2 gene is knocked out, inserted and subjected to base mutation by using the CRISPR / Cas9 technology in the Vero81 cells is proposed for the first time, a brand new concept is provided for cytology or genetic research, and the application prospect is very wide; and according to the Vero cell line YTHDF2 gene modification method provided by the invention, the mRNA methylation level in host cells can be changed, so that the virus multiplication capacity is changed, and the possibility is provided for researching the relationship between the virus and host methylation.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an m6A "reader" YTHDF2 gene modification method and its application. Background technique [0002] There are more than 150 kinds of modifications in RNA, mainly including N6-methyladenosine (N6-methyladenosine, m6A), 5-methylcytidine (m5C), inosine (I), pseudouridine (Ψ), Modifications such as N1-methylated adenosine (m1A) and 5-hydroxymethylcytidine (hm5C) are found in eukaryotic mRNA and can affect the metabolism and function of mRNA. N6-methyladenosine (m6A) is the most common post-transcriptional base modification on eukaryotic mRNA, with a frequency of about 3-5 residues per mRNA, and more than 80% of RNA bases in eukaryotic cells are methylated It is m6A modification (Zhao BS, Roundtree IA, He C. Post-transcriptional gene regulation by mRNA modifications, Nat Rev Mol Cell Biol, 2017, 18(1): 31-42). As early as the 1970s, m6A modification has been found in euka...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/55C12N15/12C12N5/10C12N7/00C12R1/93
CPCC12N15/85C12N9/22C07K14/47C12N5/0686C12N7/00C12N2510/00C12N2770/20051
Inventor 唐玉新宋德平张帆帆叶昱黄冬艳
Owner JIANGXI AGRICULTURAL UNIVERSITY
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