CN112941105A - Gene modification method of YTHDF2 of m6A 'reader' and application thereof - Google Patents
Gene modification method of YTHDF2 of m6A 'reader' and application thereof Download PDFInfo
- Publication number
- CN112941105A CN112941105A CN202110180255.0A CN202110180255A CN112941105A CN 112941105 A CN112941105 A CN 112941105A CN 202110180255 A CN202110180255 A CN 202110180255A CN 112941105 A CN112941105 A CN 112941105A
- Authority
- CN
- China
- Prior art keywords
- ythdf2
- gene
- reader
- genetic modification
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 17
- 238000012239 gene modification Methods 0.000 title claims abstract description 15
- 101000744745 Homo sapiens YTH domain-containing family protein 2 Proteins 0.000 title claims description 27
- 102100039644 YTH domain-containing family protein 2 Human genes 0.000 title claims description 26
- 210000004027 cell Anatomy 0.000 claims abstract description 59
- 239000013612 plasmid Substances 0.000 claims abstract description 35
- 101150039296 ythdf2 gene Proteins 0.000 claims abstract description 30
- 238000005516 engineering process Methods 0.000 claims abstract description 18
- 108091033409 CRISPR Proteins 0.000 claims abstract description 17
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 12
- 238000010276 construction Methods 0.000 claims abstract description 12
- 238000010362 genome editing Methods 0.000 claims abstract description 9
- 238000010354 CRISPR gene editing Methods 0.000 claims abstract description 8
- 238000003144 genetic modification method Methods 0.000 claims abstract description 8
- 238000013461 design Methods 0.000 claims abstract description 3
- 238000012216 screening Methods 0.000 claims abstract description 3
- 230000005017 genetic modification Effects 0.000 claims abstract 4
- 235000013617 genetically modified food Nutrition 0.000 claims abstract 4
- 238000012795 verification Methods 0.000 claims description 9
- 108091026890 Coding region Proteins 0.000 claims description 8
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 claims description 8
- 238000000137 annealing Methods 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 5
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 5
- 238000003209 gene knockout Methods 0.000 claims description 5
- 230000035755 proliferation Effects 0.000 claims description 5
- 210000004962 mammalian cell Anatomy 0.000 claims description 4
- 230000009466 transformation Effects 0.000 claims description 4
- 241000282552 Chlorocebus aethiops Species 0.000 claims description 3
- 238000011426 transformation method Methods 0.000 claims description 3
- 241000238631 Hexapoda Species 0.000 claims description 2
- 230000002068 genetic effect Effects 0.000 claims description 2
- 210000005253 yeast cell Anatomy 0.000 claims description 2
- 210000003292 kidney cell Anatomy 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 16
- 230000011987 methylation Effects 0.000 abstract description 9
- 238000007069 methylation reaction Methods 0.000 abstract description 9
- 241000700605 Viruses Species 0.000 abstract description 8
- 210000003501 vero cell Anatomy 0.000 abstract description 5
- 230000005727 virus proliferation Effects 0.000 abstract description 4
- 230000008859 change Effects 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 230000004170 mRNA methylation Effects 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 16
- 230000004048 modification Effects 0.000 description 15
- 238000012986 modification Methods 0.000 description 15
- 108020004999 messenger RNA Proteins 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 101100214180 Drosophila melanogaster Ythdf gene Proteins 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 6
- 238000001976 enzyme digestion Methods 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 230000033228 biological regulation Effects 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 230000029812 viral genome replication Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000867607 Chlorocebus sabaeus Species 0.000 description 2
- 239000012880 LB liquid culture medium Substances 0.000 description 2
- 239000012097 Lipofectamine 2000 Substances 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108020000999 Viral RNA Proteins 0.000 description 2
- 150000003838 adenosines Chemical class 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000005138 cryopreservation Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 230000001124 posttranscriptional effect Effects 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000012772 sequence design Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- NFEXJLMYXXIWPI-JXOAFFINSA-N 5-Hydroxymethylcytidine Chemical compound C1=C(CO)C(N)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NFEXJLMYXXIWPI-JXOAFFINSA-N 0.000 description 1
- ZAYHVCMSTBRABG-UHFFFAOYSA-N 5-Methylcytidine Natural products O=C1N=C(N)C(C)=CN1C1C(O)C(O)C(CO)O1 ZAYHVCMSTBRABG-UHFFFAOYSA-N 0.000 description 1
- ZAYHVCMSTBRABG-JXOAFFINSA-N 5-methylcytidine Chemical compound O=C1N=C(N)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 ZAYHVCMSTBRABG-JXOAFFINSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 241000710781 Flaviviridae Species 0.000 description 1
- 208000031886 HIV Infections Diseases 0.000 description 1
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 108020005198 Long Noncoding RNA Proteins 0.000 description 1
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 229930185560 Pseudouridine Natural products 0.000 description 1
- PTJWIQPHWPFNBW-UHFFFAOYSA-N Pseudouridine C Natural products OC1C(O)C(CO)OC1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-UHFFFAOYSA-N 0.000 description 1
- 230000006093 RNA methylation Effects 0.000 description 1
- 230000026279 RNA modification Effects 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- 102000004357 Transferases Human genes 0.000 description 1
- 108090000992 Transferases Proteins 0.000 description 1
- 101150100755 Ythdf gene Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WGDUUQDYDIIBKT-UHFFFAOYSA-N beta-Pseudouridine Natural products OC1OC(CN2C=CC(=O)NC2=O)C(O)C1O WGDUUQDYDIIBKT-UHFFFAOYSA-N 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000002380 cytological effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000008642 heat stress Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 230000017156 mRNA modification Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- PTJWIQPHWPFNBW-GBNDHIKLSA-N pseudouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-GBNDHIKLSA-N 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 102000018477 tRNA Methyltransferases Human genes 0.000 description 1
- 108010066587 tRNA Methyltransferases Proteins 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- -1 uniformly blowing Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20051—Methods of production or purification of viral material
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Urology & Nephrology (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Virology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供一种m6A“阅读器”YTHDF2基因改造方法及其应用，涉及基因工程技术领域。本发明的基因改造方法利用基因编辑技术对YTHDF2基因进行改造，获得敲除YTHDF2基因的细胞系，包括：sgRNA序列的设计，YTHDF2基因敲除，质粒构建和改造基因重组细胞系筛选。本发明对具有特异性基因的细胞进行基因改造，并首次提出在Vero81细胞中使用CRISPR/Cas9技术对YTHDF2基因进行敲除、插入、碱基突变，为细胞学或基因学研究提供了全新的概念，具有十分广泛的应用前景；本发明提供的Vero细胞系YTHDF2基因改造方法能够改变宿主细胞中mRNA甲基化水平，从而改变病毒增殖能力，为研究病毒与宿主甲基化的关系提供可能。
The invention provides an m6A "reader" YTHDF2 gene modification method and application thereof, and relates to the technical field of genetic engineering. The genetic modification method of the invention utilizes gene editing technology to modify the YTHDF2 gene to obtain a cell line that knocks out the YTHDF2 gene, including: the design of sgRNA sequences, the knockout of the YTHDF2 gene, the construction of plasmids and the screening of recombinant cell lines with modified genes. The invention carries out genetic modification on cells with specific genes, and firstly proposes to use CRISPR/Cas9 technology to knock out, insert, and mutate the YTHDF2 gene in Vero81 cells, which provides a new concept for cytology or genetics research. , has a very wide application prospect; the Vero cell line YTHDF2 gene modification method provided by the present invention can change the mRNA methylation level in the host cell, thereby changing the virus proliferation ability, and providing the possibility to study the relationship between the virus and the host methylation.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a YTHDF2 genetic modification method of an m6A 'reader' and application thereof.
Background
The RNA has more than 150 modifications, mainly comprising N6-methylated adenosine (N6-methylaldenosine, m6A), 5-methylcytidine (m5C), inosine (I), pseudouridine (psi), N1-methylated adenosine (m1A), 5-hydroxymethylcytidine (hm5C) and the like, and the modifications are found in eukaryotic mRNA and can influence the metabolism and function of the mRNA. N6-methyaddenesine (m6A) is the most common posttranscriptional base modification on eukaryotic mRNA, occurring at about 3-5 residues per mRNA, and more than 80% of the RNA base methylation in eukaryotic cells is the 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). The m6A modification has been found in eukaryotic mRNA and lncRNA in the early 70 s. Methylation modification of the mRNA 5' UTR region in most eukaryotes is known to play an important role in mRNA splicing, editing, stability, degradation, polyadenylation, etc.; while methylation modification in the 3 'UTR region contributes to the out-of-core transport, translation initiation of mRNA and maintenance of mRNA structural stability with polyA binding protein (Fu Y, dominisini D, Rechavi G, He C. Gene expression programmed through conversion M (6) A RNAmethylation. Nat Rev Genet,2014,15(5): 293. 306; Nandan S. Gokhale, et al. RNA modifications in the genome, Plos Pathologens, 2017,1006188; Schwartz S, Mumbach MR, Jovanovic M, et al. Perturbation of M6A precursors in the expression of mRNA molecules and translation of internal 5' sites. 296, 2014, 284 (1): 284). The recognition site of most RNA methylases is RRACH, also reported as [ G/A/U ] [ G > A ] m6AC [ U > A > C ]. The m6A methylation modification is proved to be reversible, and is participated by methylation transferase (Writers), demethylase (Erasers) and methylation reading protein (Readers). It has been found that many m6A modification sites (Lichinchi G, et al. dynamics of the human and viral m (6) A RNA genes along HIV-1infection of T cells. Nature microbiology.2016; 1(4):16011) exist on viral RNA, but it is not clear how the modification of m6A affects viral replication.
Recent studies have shown that YTHDF is a reading protein of m6A, and binding of YTHDF alters the translation efficiency and stability of m6A RNA (Wang X, et al, N6-methylenedenosine-dependent regulation of messenger RNA stability. Nature,2014,505,117-120. Du H, et al, YTHDF2 stabilizers m (6) A-dependent RNA direct regulation of the CCR4-NOT dependent enzyme complex. Nat. Commun.2016,7,12626.). However, it is not known how the YTHDF protein affects the proliferation of the virus through m6A during the infection of cells by porcine epidemic diarrhea virus (Nandan S. Gokhale, et al. N6-Methyldenosine in Flaviviridae Viral RNA genes infection. cell Host Microbe.2016,20 (654) (5): -. Studies have shown that the YTHDF protein can recognize HCV RNA and negatively regulate HCV progeny virus production. Since HCV viruses are replicated mainly in intracellular liposomes, the YTHDF proteins in HCV-infected cells are localized by immunofluorescence technology, and HCV infection is found to induce localization of YTHDF proteins to liposomes, recognize and combine with HCV RNA, playing an important role in the life cycle of the viruses.
Disclosure of Invention
The invention aims to provide a method for modifying YTHDF2 gene of an m6A 'reader' and application thereof, wherein the characteristic that YTHDF gene can recognize virus RNA and negatively regulate virus proliferation is utilized, and the methylation level of mRNA in host cells can be changed by modifying Vero cell line YTHDF2 gene, so that the virus replication capacity is changed.
In order to achieve the purpose, the invention provides a method for modifying YTHDF2 gene of m6A 'reader', wherein YTHDF2 gene is modified by using gene editing technology to obtain a cell line with YTHDF2 gene knocked out, which comprises the following steps: sgRNA sequence design, YTHDF2 gene knockout, plasmid construction and screening of modified gene recombinant cell lines.
For the technical solutions mentioned above, further, the gene editing technology is CRISPR/Cas9 technology.
The invention uses CRISPR/Cas9 gene editing technology and Vero81 cell line as an example for transformation, and carries out mutation transformation on YTHDF2 gene in the genome. YTHDF2 is a recently identified protein that can act as a "reader" that specifically recognizes the m6A modification site on transcripts. After recognizing the m6A modification site, YTHDF2 can participate in the regulation and control of a series of physiological or pathological processes through the regulation of the stability of target RNA. However, it should be noted that the CRISPR/Cas9 gene editing technology is used for the above transformation, but the CRISPR/Cas9 technology is not used for the gene editing technology, and the existing gene editing technologies such as TALEN technology and ZFN technology are all applicable.
In the above technical solution, further, the sgRNA sequence is designed by selecting the front 1/3 regions of the third coding region and the fourth coding region of the m6A "reader" YTHDF2 gene, and the sequences are shown as SEQ ID No.3 and SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, and SEQ ID No.7 and SEQ ID No. 8. The m6A 'reader' YTHDF2 gene sequence is originated from Vero cell YTHDF2 gene, and YTHDF2 nucleotide sequence and protein sequence are shown in SEQ ID NO.1 and SEQ ID NO. 2.
In the above technical solution, further, the YTHDF2 gene knockout technical method further comprises an annealing system: sgRNA-F (100. mu.M) 1. mu. L, sgRNA-R (100. mu.M) 1. mu.L, 10 XT 4 Ligation Buffer 1. mu. L, ddH2O 6.5μL、T4 PNK 0.5μL。
In the above technical scheme, further, the plasmid construction comprises recombinant plasmid construction and verification plasmid construction, wherein the recombinant plasmid is pCas9-YTHDF2-1, pCas9-YTHDF2-2, pCas9-YTHDF2-3, and the verification plasmid is pCDNA3.1-mcherry-YTHDF 2.
In the above-described embodiments, further, the recombinant cell line is a eukaryotic cell.
For the above technical solution, further, the eukaryotic cell is any one of a mammalian cell, a yeast cell, and an insect cell.
In the above technical solution, further, the mammalian cell is selected from Vero 81.
In the technical scheme, the screened recombinant cell line is Vero81-YTHDF 2-KD.
According to the invention, a Cas9 recombinant plasmid is constructed through sgRNA, and a knockout recombinant cell line with high-efficiency cutting performance on YTHDF2 gene is screened out after cells are transfected. The modification is verified on HEK 293T cells and modified on Vero81 cells, but the invention does not show that the modification can only be applied to Vero81 cell lines and is applicable to other cell lines.
The second purpose of the invention is to provide an application of the gene modification method, in particular to an application of the gene modification method in changing the porcine epidemic diarrhea virus proliferation.
The invention has the beneficial effects that:
the invention carries out gene modification on cells with specific genes, and then eliminates the cells after the gene modification by utilizing targeted methods such as targeted drugs, immunotherapy and the like. The invention provides the first time that the YTHDF2 gene is knocked out, inserted and subjected to base mutation by using the CRISPR/Cas9 technology in the Vero81 cell, provides a brand-new concept for cytological or genetic research, and has a very wide application prospect.
The Vero cell line YTHDF2 gene modification method provided by the invention can change the mRNA methylation level in host cells, thereby changing the virus multiplication capacity and providing possibility for researching the relation between viruses and host methylation.
Drawings
FIG. 1 is a schematic diagram of PCR identification of a bacterial liquid of the recombinant plasmid pCas9-YTHDF2 of the present invention; 1-15: PCR products of bacterial liquid; 16: negative control; m: DNA Marker DL 2000;
FIG. 2 is a schematic diagram showing the double digestion results of the PCR products of pCDNA3.1-mcherry and YTHDF2 of the present invention; 1: the double digestion result of pCDNA3.1-mcherry plasmid; 2: double enzyme digestion result of YTHDF2 PCR product; m: DNA Marker DL 15000;
FIG. 3 is a schematic diagram of PCR identification of the bacterial liquid of the recombinant plasmid pCDNA3.1-mcherry-YTHDF2 of the present invention; 1-5: PCR products of bacterial liquid; m: DNAmarker DL 2000;
FIG. 4 is a pX459 plasmid map according to the present invention;
FIG. 5 is a map of the pCDNA3.1-mcherry plasmid of the present invention;
FIG. 6 is a schematic diagram showing the cutting microscopic fluorescence intensity of the recombinant plasmid pCas9-YTHDF2 sgRNA of the present invention;
A：pCas9-control+pCDNA3.1-mcherry-YTHDF2；
B：pCas9-YTHDF2-1+pCDNA3.1-mcherry-YTHDF2；
C：pCas9-YTHDF2-2+pCDNA3.1-mcherry-YTHDF2；
D：pCas9-YTHDF2-3+pCDNA3.1-mcherry-YTHDF2；
FIG. 7 is a graph showing the effect of wild-type and mutant cells of the present invention on the proliferation of porcine epidemic diarrhea virus.
The invention is illustrated by the following examples, which are intended to be illustrative only and not to be limiting in any way. The specific experimental procedures not mentioned in the examples are usually carried out according to conventional or obvious experimental procedures.
The invention is further explained with reference to the drawings and the embodiments.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples are given without reference to specific techniques or conditions, as described in the literature or in the literature of the art (for example, see J. SammBruk et al, molecular cloning, A Laboratory Manual (fourth edition), Huang Petang et al, science publishers; Jennifer Doudna, prashan Manual, CRISRP-Cas: Alabor manual, Cold Spring Harbor Laboratory Press) or according to product specifications. The reagents or instruments used are not indicated to the manufacturer, and are all conventional products purchased through provincial bidding purchasing departments.
The above-mentioned technical features of the present invention and those described in detail below (e.g., in the embodiments) can be combined with each other to form a new or preferred embodiment.
BLAST alignment analysis was performed based on the YTHDF2 gene sequence of African green monkey (Chlorocebus sabaeus), and the gene number of YTHDF2 in Vero81 was determined to be 103225249. The nucleotide sequence of the code YTHDF2 is shown as SEQ ID NO.1, and the protein sequence of YTHDF2 is shown as SEQ ID NO. 2.
Example 1: CRISPR/Cas9 modified Vero81 cell line YTHDF2 protein
The cell gene modification method of the embodiment of the invention comprises the following steps:
1. according to coding regions and non-coding regions of YTHDF2 gene sequences of African green monkeys (Chlorocebus sabaeus), after comprehensive alignment, selecting a front end 1/3 region of a third coding region and a fourth coding region for sgRNA sequence design, and selecting three sgRNAs according to a sgRNA design principle for efficiency verification. The sgRNA in this example was selected on the basis of moderate GC content (40% -60%), with the selected region located at the front end of the gene.
In this example, the sgRNA sequence of YTHDF2 is as follows:
sgRNA1-F (SEQ ID NO. 3): 5'-CACCGTCTTATGGACAACTGAGCAA-3' and sgRNA1-R (SEQ ID NO. 4): 5'-CTTGCTCAGTTGTCCATAAGACAAA-3', respectively;
sgRNA2-F (SEQ ID NO. 5): 5'-CACCGAGACGAAGAATGGCATTGCA-3' and sgRNA2-R (SEQ ID NO. 6): 5'-CTGCAATGCCATTCTTCGTCTCAAA-3', respectively;
sgRNA3-F (SEQ ID No.): 5'-CACCGGTCCATTACTAGTAACATCG-3' and sgRNA3-R (SEQ ID NO. 8): 5'-CCGATGTTACTAGTAATGGACCAAA-3' are provided.
2. The plasmid construction and transformation method is as follows: annealing was performed using the oligosaccharide nucleotide chains sgRNA-F and sgRNA-R.
The annealing system is: sgRNA-F (100. mu.M) 1. mu. L, sgRNA-R (100. mu.M) 1. mu.L, 10 XT 4 Ligation Buffer 1. mu. L, ddH2O 6.5μL、T4 PNK 0.5μL。
Annealing was performed according to the following annealing program: 30min at 37 ℃ and 30min at 95 ℃, and then uniformly reducing the temperature to 25 ℃ at 5 ℃/min.
The plasmid pX459 (FIG. 4) was used as a template for digestion. Taking 50 mu g of enzyme digestion product, 1 mu L of annealing product (diluted by 1: 200), 5 mu L of 2 XQuickligation Buffer and 1 mu L of Quick Ligase, and supplementing ddH2O to 10. mu.L. After mixing uniformly, connecting for 10min at room temperature, adding 5 μ L of the connecting product into 50 μ L of Top10 competent cells, incubating on ice for 30min, placing on ice again for incubation for 2-3min after heat stress at 42 ℃ for 90s, adding 950 μ L of LB liquid culture medium, placing in a shaking incubator at 37 ℃ and 200rpm for culturing for 90min, centrifuging at 8000rpm for 5min, discarding 800 μ L of supernatant, uniformly blowing, coating in an Amp-resistant LB solid culture medium, and placing in a constant temperature incubator at 37 ℃ for culturing for 14-18 h. Respectively picking 15 monoclonals to shake in LB liquid culture medium with Amp, identifying bacterial liquid PCR as positive and sequencing to determine correct, extracting plasmid with endotoxin-free kit, subpackaging and storing, and selecting three recombinant plasmids named pCas9-YTHDF2-1, pCas9-YTHDF2-2 and pCas9-YTHDF2-3, wherein the PCR identification result of bacterial liquid of recombinant plasmid pCas9-YTHDF2 is shown in figure 1.
3. And verifying the construction and amplification of the DNA plasmid.
To facilitate verification of the cleavage efficiency of sgrnas, the present embodiment inserts the donor DNA into pcdna3.1-mcherry plasmid (fig. 5) to obtain a verification recombinant plasmid.
4. Construction of the verification plasmid pCDNA3.1-mcherry-YTHDF 2.
Using Vero81 cell genome DNA as a template, and using a primer YTHDF2-F (SEQ ID NO. 9): 5'-CGGGATCCAATAATGCGTATACTGCCATGTC-3' and YTHDF2-R (SEQ ID NO. 10): 5'-GGAATTCCACT TTCTTCTTCCTCTTGGCG-3' PCR amplification is carried out, and the amplified product is recovered by gel. Carrying out BamHI and EcoRI double enzyme digestion on the PCR gel recovery product and the pCDNA3.1-mcherry plasmid respectively, taking 20 mu L of the gel recovery product and the plasmid respectively, adding BamHI2.5 mu L, EcoRI 2.5 mu L and 10 XK buffer 5 mu L respectively, adding water to complement to 50 mu L, mixing uniformly, carrying out water bath at 37 ℃ for 4h, obtaining the enzyme digestion result as shown in figure 2, and obtaining the enzyme digestion product after gel recovery. The two enzyme digestion products are respectively added with 2 mu L, T4 ligase and 1 mu L, T4 ligase buffer solution in 1 mu L, water is added to make up to 10 mu L, and the mixture is uniformly mixed and then placed in a refrigerator at 4 ℃ for overnight connection. The ligation product is transformed, spotted, sequenced and identified, and then the plasmid is extracted and stored for later use, named as pCDNA3.1-mcherry-YTHDF2, and the PCR identification result is shown in figure 3.
Example 2: modified Vero81 cell YTHDF2 protein expression detection
1. The cutting efficiency of the pCas9-YTHDF2 plasmid is verified.
Taking out Vero81 cells from a liquid nitrogen tank, rapidly placing the Vero81 cells in a flowing water bath kettle at 37 ℃, wiping the outside of a cryopreservation tube with alcohol cotton on a cell room super clean bench after thawing completely for about 2-3min, transferring the frozen cryopreservation tube to a 25mL cell culture bottle, replacing a corresponding fresh complete culture medium after the cells adhere to the wall for 4h, and carrying out 1:3 passage on the cells after the cells are overgrown. The Vero81 cells are inoculated into a 6-hole cell culture plate for culture 24 hours before the experiment until the cell density reaches 70-80% of the confluence rate. The plasmids pCas9-YTHDF2-1, pCas9-YTHDF2-2, pCas9-YTHDF2-3 and pCDNA3.1-mcherry-YTHDF2 were removed and the nucleic acid concentration was determined using NanoDrop 2000. Mixing a transfection reagent Lipofectamine 2000, pCas9-YTHDF2 and pCDNA3.1-mcherry-YTHDF2 according to a certain proportion, adding a serum-free culture medium with a proper volume, standing the mixed solution at room temperature for 10-15min, adding a six-well plate, changing the mixed solution into a 10% FBS DMEM high-sugar medium for 4-6h, and photographing by using a fluorescence microscope to calculate the fluorescence intensity after culturing for 24-48 h. The results are shown in FIG. 6, which shows that pCas9-YTHDF2-1 has the highest cleavage efficiency and was selected as the material for the next stage.
2. Transfection and selection of Vero81 cells.
Plating Vero81 cells one day before experiment to prepare transfection; on the day of the experiment, the cells in the previous day are taken out from the cell culture box and observed under a microscope to achieve 70-80% of confluence rate, and then transfection can be carried out. The pCas9-YTHDF2-1 plasmid and Lipofectamine 2000 were used for transfection, puromycin with a final concentration of 2ng/mL was added 24h after transfection for selection, and the monoclonal cell lines were selected by the infinite multiple dilution method after 9-10 d.
3. The knockout result of YTHDF2 gene of Vero81 cells is verified.
Extracting genome DNA of the monoclonal cell strain, taking Vero81 cell genome DNA as a template, and carrying out extraction by using a primer YTHDF2-F (SEQ ID NO. 9): 5'-CGGGATCCAATAATGCGTATACTG CCATGTC-3' and YTHDF2-R (SEQ ID NO. 10): 5'-GGAATTCCACTTTCTTCTTC CTCTTGGCG-3' PCR amplification is carried out, and the amplified product is subjected to gel recovery sequencing. The cell line with the sequencing result of the encoded protein being shifted and in good growth state was selected as the result for verification and named Vero81-YTHDF2-KD cell line.
Example 3: effect of modified Vero cell YTHDF2 protein on Virus proliferation
The YTHDF2 gene knockout can specifically influence the proliferation of Porcine Epidemic Diarrhea Virus (PEDV). And comparing the expression quantity of YTHDF2 genes in the expression quantity of the N genes of the porcine epidemic diarrhea viruses in the wild Vero81 cells and the modified Vero81 cells by real-time fluorescent quantitative PCR amplification. As can be seen from FIG. 7, compared with the wild Vero81 cell, the modified Vero81-YTHDF2-KD cell of the porcine epidemic diarrhea virus has a higher proliferation effect than that of the wild Vero81-YTHDF2-KD cell. Thus, knockout of YTHDF2 significantly promoted viral replication.
Finally, it should be emphasized that the above-described preferred embodiments of the present invention are merely examples of implementations, rather than limitations, and that many variations and modifications of the invention are possible to those skilled in the art, without departing from the spirit and scope of the invention.
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110180255.0A CN112941105A (en) | 2021-02-08 | 2021-02-08 | Gene modification method of YTHDF2 of m6A 'reader' and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110180255.0A CN112941105A (en) | 2021-02-08 | 2021-02-08 | Gene modification method of YTHDF2 of m6A 'reader' and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112941105A true CN112941105A (en) | 2021-06-11 |
Family
ID=76245048
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110180255.0A Pending CN112941105A (en) | 2021-02-08 | 2021-02-08 | Gene modification method of YTHDF2 of m6A 'reader' and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112941105A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115029357A (en) * | 2022-06-21 | 2022-09-09 | 首都师范大学 | dsRNA of insect m6A methylation reading protein elF3-S6 gene and its application |
| CN120098934A (en) * | 2025-03-07 | 2025-06-06 | 江西省农业科学院畜牧兽医研究所 | A porcine IPO4 gene knockout cell line and its construction method and application |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170067021A1 (en) * | 2015-07-31 | 2017-03-09 | Regents Of The University Of Minnesota | Modified cells and methods of therapy |
| US20190100761A1 (en) * | 2016-04-06 | 2019-04-04 | Duke University | Compositions and methods for enhanced gene expression and viral replication |
| WO2019074980A1 (en) * | 2017-10-09 | 2019-04-18 | Stowers Institute For Medical Research | Methods and compositions for expansion of cell population |
| CN109680064A (en) * | 2019-01-17 | 2019-04-26 | 深圳市罗湖区人民医院 | Application of the YTHDF2 gene in bladder transitional cell carcinoma is diagnosed, prevented and treated |
| CN110423785A (en) * | 2019-05-29 | 2019-11-08 | 扬州大学 | A kind of cell line and its construction method of the knockout chicken KPNA3 gene based on CRISPR-Cas9 editing technique |
| US20190374576A1 (en) * | 2016-10-27 | 2019-12-12 | Intima Bioscience, Inc. | Viral methods of t cell therapy |
| WO2020081937A1 (en) * | 2018-10-19 | 2020-04-23 | The University Of Chicago | Methods and compositions for treating negative-sense single-stranded rna virus |
| CN111549059A (en) * | 2020-04-30 | 2020-08-18 | 中国农业科学院兰州兽医研究所 | TPL2 gene knockout HEK293T cell line and its construction method and application |
| CN111849979A (en) * | 2020-06-22 | 2020-10-30 | 中国农业科学院兰州兽医研究所 | A kind of sgRNA targeting knockout of RPSA gene and construction method of RPSA gene knockout cell line |
| US20200377617A1 (en) * | 2019-05-29 | 2020-12-03 | Duke University | Compositions and methods for detection and measurement of rna modifications through targeted rna editing |
| CN112513250A (en) * | 2018-04-20 | 2021-03-16 | 加利福尼亚大学董事会 | Fusion proteins and fusion ribonucleic acids for tracking and manipulating cellular RNA |
| CN112791094A (en) * | 2021-01-13 | 2021-05-14 | 南京医科大学 | Substances that down-regulate YTHDF2 protein expression and their applications |
| US20210154236A1 (en) * | 2018-03-29 | 2021-05-27 | The University Court Of The University Of Edinburgh | Haematopoietic stem cell treatment |
| WO2022086935A1 (en) * | 2020-10-19 | 2022-04-28 | The General Hospital Corporation | Targeting xist and rna methylation for x reactivation therapy |
-
2021
- 2021-02-08 CN CN202110180255.0A patent/CN112941105A/en active Pending
Patent Citations (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20170067021A1 (en) * | 2015-07-31 | 2017-03-09 | Regents Of The University Of Minnesota | Modified cells and methods of therapy |
| US20190100761A1 (en) * | 2016-04-06 | 2019-04-04 | Duke University | Compositions and methods for enhanced gene expression and viral replication |
| US20190374576A1 (en) * | 2016-10-27 | 2019-12-12 | Intima Bioscience, Inc. | Viral methods of t cell therapy |
| WO2019074980A1 (en) * | 2017-10-09 | 2019-04-18 | Stowers Institute For Medical Research | Methods and compositions for expansion of cell population |
| US20200370044A1 (en) * | 2017-10-09 | 2020-11-26 | Stowers Institute For Medical Research | Methods and compositions for expansion of cell population |
| US20210154236A1 (en) * | 2018-03-29 | 2021-05-27 | The University Court Of The University Of Edinburgh | Haematopoietic stem cell treatment |
| CN112867789A (en) * | 2018-03-29 | 2021-05-28 | 爱丁堡大学董事会 | hematopoietic stem cell therapy |
| CN112513250A (en) * | 2018-04-20 | 2021-03-16 | 加利福尼亚大学董事会 | Fusion proteins and fusion ribonucleic acids for tracking and manipulating cellular RNA |
| WO2020081937A1 (en) * | 2018-10-19 | 2020-04-23 | The University Of Chicago | Methods and compositions for treating negative-sense single-stranded rna virus |
| US20220033783A1 (en) * | 2018-10-19 | 2022-02-03 | The University Of Chicago | Methods and compositions for treating negative-sense single-stranded rna virus |
| CN109680064A (en) * | 2019-01-17 | 2019-04-26 | 深圳市罗湖区人民医院 | Application of the YTHDF2 gene in bladder transitional cell carcinoma is diagnosed, prevented and treated |
| US20200377617A1 (en) * | 2019-05-29 | 2020-12-03 | Duke University | Compositions and methods for detection and measurement of rna modifications through targeted rna editing |
| CN110423785A (en) * | 2019-05-29 | 2019-11-08 | 扬州大学 | A kind of cell line and its construction method of the knockout chicken KPNA3 gene based on CRISPR-Cas9 editing technique |
| CN111549059A (en) * | 2020-04-30 | 2020-08-18 | 中国农业科学院兰州兽医研究所 | TPL2 gene knockout HEK293T cell line and its construction method and application |
| CN111849979A (en) * | 2020-06-22 | 2020-10-30 | 中国农业科学院兰州兽医研究所 | A kind of sgRNA targeting knockout of RPSA gene and construction method of RPSA gene knockout cell line |
| WO2022086935A1 (en) * | 2020-10-19 | 2022-04-28 | The General Hospital Corporation | Targeting xist and rna methylation for x reactivation therapy |
| CN112791094A (en) * | 2021-01-13 | 2021-05-14 | 南京医科大学 | Substances that down-regulate YTHDF2 protein expression and their applications |
Non-Patent Citations (6)
| Title |
|---|
| JIANING CHEN等: "N6-methyladenosine regulates PEDV replication and host gene expression", 《VIROLOGY》, vol. 548, 16 June 2020 (2020-06-16), pages 64 * |
| 吉春苗: "猪流行性腹泻病毒感染、宿主抗体库应答及m6A修饰调控的研究", 《中国博士学位论文全文数据库基础科学辑》 * |
| 吉春苗: "猪流行性腹泻病毒感染、宿主抗体库应答及m6A修饰调控的研究", 《中国博士学位论文全文数据库基础科学辑》, 15 January 2022 (2022-01-15), pages 006 - 318 * |
| 唐朝晖主编: "《生命科学综合设计实验指南》", 华中科技大学出版社, pages: 121 - 131 * |
| 巩沅鑫等: "病毒基因组中m~6A甲基化修饰调控机制及研究进展", 《病毒学报》 * |
| 巩沅鑫等: "病毒基因组中m~6A甲基化修饰调控机制及研究进展", 《病毒学报》, no. 05, 14 August 2018 (2018-08-14), pages 6 - 12 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115029357A (en) * | 2022-06-21 | 2022-09-09 | 首都师范大学 | dsRNA of insect m6A methylation reading protein elF3-S6 gene and its application |
| CN115029357B (en) * | 2022-06-21 | 2023-05-09 | 首都师范大学 | dsRNA of insect m6A methylation reading protein eIF3-S6 gene and its application |
| CN120098934A (en) * | 2025-03-07 | 2025-06-06 | 江西省农业科学院畜牧兽医研究所 | A porcine IPO4 gene knockout cell line and its construction method and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107502608B (en) | Construction method and application of sgRNA for knocking out human ALDH2 gene and ALDH2 gene deletion cell line | |
| US11898270B2 (en) | Pig genome-wide specific sgRNA library, preparation method therefor and application thereof | |
| CN109880851B (en) | Screening reporter vector and screening method for enriching CRISPR/Cas9-mediated homologous recombination repair cells | |
| CN106222177A (en) | The CRISPR Cas9 system of a kind of targeted human STAT6 and for treating the application of anaphylactic disease | |
| CN107893073B (en) | Method for screening glutamine synthetase defect type HEK293 cell strain | |
| Fehr | Bacterial artificial chromosome-based lambda red recombination with the I-SceI homing endonuclease for genetic alteration of MERS-CoV | |
| CN112941105A (en) | Gene modification method of YTHDF2 of m6A 'reader' and application thereof | |
| Vopálenský et al. | Messenger RNAs of yeast virus-like elements contain non-templated 5′ Poly (A) leaders, and their expression is independent of eIF4E and Pab1 | |
| CN119219794B (en) | OpenCRISPR-NLS fusion protein for gene editing of penaeus monodon and application of OpenCRISPR-NLS fusion protein | |
| CN110499316B (en) | Milk cow mammary gland epithelial cell line with CD44 gene knocked out and construction method thereof | |
| CN113151277A (en) | Construction method of chicken DF-1 cell IHH gene knockout stable cell strain and specific sgRNA thereof | |
| CN106282224A (en) | Method and the eukaryotic cell of artificial chromosome is built in target cell | |
| CN117363644A (en) | VIGS silencing efficiency reporting plasmid, method for evaluating silencing efficiency and application | |
| CN110144626A (en) | A kind of construction method of promoter library | |
| CN116064541A (en) | A method for knocking out pig cell Y chromosome based on CRISPR/Cas9 system | |
| CN117467617B (en) | MA-104 cell line for knocking out vimentin gene, construction method and application thereof | |
| CN101532181A (en) | Method and kit for in-situ construction of gene mutation library | |
| CN107058302B (en) | Targeted Knockout of Nrf1β Gene Recognition Sequence Pair, Talens, Vector Pair and Application in Human Hepatocytes | |
| CN118853659A (en) | Nucleic acid for increasing the output of mRNA translation protein and its application | |
| CN121380358A (en) | Application of African swine fever virus host restriction factor PML in disease-resistant breeding screening | |
| CN121046383A (en) | Enhancers that inhibit C2C12 cell differentiation and their applications | |
| CN120700049A (en) | A DF-1 cell line with miR-155 gene deletion constructed based on CRISPR/Cas9 technology | |
| CN115851715A (en) | Buffalo skeletal muscle specific high-efficiency promoter as well as screening method and application thereof | |
| CN120866301A (en) | Plasmodium multiplex nucleic acid detection kit and matched artificial chromosome engineering bacteria quality control product | |
| CN115433736A (en) | Gateway prokaryotic vector system for high-efficiency expression and purification of small tag active fusion proteins |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |