CN114934068B - Alkbh5基因稳定敲除的小鼠卵巢颗粒细胞株及构建方法和应用 - Google Patents
Alkbh5基因稳定敲除的小鼠卵巢颗粒细胞株及构建方法和应用 Download PDFInfo
- Publication number
- CN114934068B CN114934068B CN202210729919.9A CN202210729919A CN114934068B CN 114934068 B CN114934068 B CN 114934068B CN 202210729919 A CN202210729919 A CN 202210729919A CN 114934068 B CN114934068 B CN 114934068B
- Authority
- CN
- China
- Prior art keywords
- alkbh5
- cells
- gene
- plasmid
- cell strain
- 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.)
- Active
Links
- 210000001672 ovary Anatomy 0.000 title claims abstract description 32
- 101150064041 ALKBH5 gene Proteins 0.000 title claims abstract description 25
- 210000004565 granule cell Anatomy 0.000 title claims abstract description 19
- 238000010276 construction Methods 0.000 title claims abstract description 13
- 238000003209 gene knockout Methods 0.000 title claims abstract description 12
- 241000699670 Mus sp. Species 0.000 title description 9
- 210000004027 cell Anatomy 0.000 claims abstract description 66
- 239000013612 plasmid Substances 0.000 claims abstract description 32
- 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 claims abstract description 30
- 201000010065 polycystic ovary syndrome Diseases 0.000 claims abstract description 18
- 239000012634 fragment Substances 0.000 claims abstract description 15
- 229950010131 puromycin Drugs 0.000 claims abstract description 15
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 13
- 238000000034 method Methods 0.000 claims abstract description 13
- 238000011160 research Methods 0.000 claims abstract description 7
- 238000012216 screening Methods 0.000 claims abstract description 5
- 230000003950 pathogenic mechanism Effects 0.000 claims abstract description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 108020004414 DNA Proteins 0.000 claims description 13
- 239000002609 medium Substances 0.000 claims description 10
- 238000000137 annealing Methods 0.000 claims description 9
- 238000012163 sequencing technique Methods 0.000 claims description 8
- 238000004113 cell culture Methods 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 7
- 210000002503 granulosa cell Anatomy 0.000 claims description 7
- 239000012096 transfection reagent Substances 0.000 claims description 7
- 239000013598 vector Substances 0.000 claims description 7
- 239000006285 cell suspension Substances 0.000 claims description 6
- 238000001890 transfection Methods 0.000 claims description 6
- 238000003776 cleavage reaction Methods 0.000 claims description 5
- 239000012737 fresh medium Substances 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 5
- 230000002441 reversible effect Effects 0.000 claims description 5
- 230000007017 scission Effects 0.000 claims description 5
- 230000000295 complement effect Effects 0.000 claims description 4
- 230000008569 process Effects 0.000 claims description 4
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- 108091000080 Phosphotransferase Proteins 0.000 claims description 3
- 229960000723 ampicillin Drugs 0.000 claims description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- 102000020233 phosphotransferase Human genes 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 230000008685 targeting Effects 0.000 claims description 3
- 102000012410 DNA Ligases Human genes 0.000 claims description 2
- 108010061982 DNA Ligases Proteins 0.000 claims description 2
- 101100378871 Mus musculus Alkbh5 gene Proteins 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims description 2
- 230000021164 cell adhesion Effects 0.000 claims 1
- 238000007865 diluting Methods 0.000 claims 1
- 230000002611 ovarian Effects 0.000 claims 1
- 101000959153 Homo sapiens RNA demethylase ALKBH5 Proteins 0.000 abstract description 16
- 102100039083 RNA demethylase ALKBH5 Human genes 0.000 abstract description 16
- 238000001976 enzyme digestion Methods 0.000 abstract description 6
- 108091033409 CRISPR Proteins 0.000 abstract description 5
- 238000013461 design Methods 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 4
- 238000010453 CRISPR/Cas method Methods 0.000 abstract description 3
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 238000012795 verification Methods 0.000 abstract description 2
- 238000003780 insertion Methods 0.000 abstract 1
- 230000037431 insertion Effects 0.000 abstract 1
- 239000002502 liposome Substances 0.000 abstract 1
- 238000012546 transfer Methods 0.000 abstract 1
- 230000001052 transient effect Effects 0.000 abstract 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 15
- 230000014509 gene expression Effects 0.000 description 8
- 230000004048 modification Effects 0.000 description 8
- 238000012986 modification Methods 0.000 description 8
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 6
- 108010082399 Autophagy-Related Proteins Proteins 0.000 description 5
- 102000005882 Histone H2a Dioxygenase AlkB Homolog 1 Human genes 0.000 description 5
- 108010005336 Histone H2a Dioxygenase AlkB Homolog 1 Proteins 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 4
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 230000008506 pathogenesis Effects 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 102000003954 Autophagy-Related Proteins Human genes 0.000 description 3
- 101100323015 Mus musculus Alk gene Proteins 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000011987 methylation Effects 0.000 description 3
- 238000007069 methylation reaction Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 102000016397 Methyltransferase Human genes 0.000 description 2
- 108060004795 Methyltransferase Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000004900 autophagic degradation Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 230000004064 dysfunction Effects 0.000 description 2
- 102000006602 glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 2
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000000865 phosphorylative effect Effects 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 101150090724 3 gene Proteins 0.000 description 1
- 208000004611 Abdominal Obesity Diseases 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 238000010354 CRISPR gene editing Methods 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010065941 Central obesity Diseases 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 201000009273 Endometriosis Diseases 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 101150027313 Has2 gene Proteins 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
- 108010085220 Multiprotein Complexes Proteins 0.000 description 1
- 101150066884 Pink1 gene Proteins 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 208000002223 abdominal aortic aneurysm Diseases 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000002513 anti-ovulatory effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000013592 cell lysate Substances 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
- 238000001816 cooling Methods 0.000 description 1
- 210000001771 cumulus cell Anatomy 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000017858 demethylation Effects 0.000 description 1
- 238000010520 demethylation reaction Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 208000030172 endocrine system disease Diseases 0.000 description 1
- 238000010201 enrichment analysis Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 201000010066 hyperandrogenism Diseases 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000021121 meiosis Effects 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 230000008182 oocyte development Effects 0.000 description 1
- 230000016087 ovulation Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 201000001245 periodontitis Diseases 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 208000017443 reproductive system disease Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
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
- 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
-
- 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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- 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
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- 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
- C12N2510/00—Genetically modified cells
- C12N2510/04—Immortalised cells
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Urology & Nephrology (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Reproductive Health (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株及构建方法和应用。本发明利用CRISPR/Cas 9相关技术，经过sgRNA的设计、插入片段的合成及酶切处理，构建出用于敲除ALKBH5的重组质粒，通过脂质体瞬转的方法，将质粒转到入小鼠卵巢颗粒细胞mGC中，经过嘌呤霉素稳转筛选以及单克隆的挑选及验证，从而建立了ALKBH5基因稳定敲除的细胞株ΔALKBH5‑mGC。本发明的细胞株可为研究ALKBH5对多囊卵巢综合症的致病机制的进一步深入研究搭建一个良好的基础。
Description
技术领域
本发明涉及一种ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株及构建方法和应用。
背景技术
多囊卵巢综合症(PCOS)作为最常见的异质性内分泌疾病之一，在全球育龄期妇女中的患病率约为6-13％，它不仅是女性无排卵性不孕的主要原因，还可增加糖尿病、血管血栓形成、子宫内膜异位症、子宫内膜癌和心血管疾病等多种疾病的风险。其诊断依据为高雄激素血症、卵巢多囊样改变和排卵功能障碍。尽管在过去的数十年间，对PCOS的治疗取得了较大的进展，但由于其高度的异质性，目前的治疗策略仍无法治愈这种疾病，因此，深入研究PCOS的病因势在必行。
卵泡是卵巢的核心功能单位，由单个卵母细胞和周围颗粒细胞、卵丘细胞和泡膜细胞组成。颗粒细胞作为卵泡内的体细胞，在卵泡发生、甾体激素生成以及生育能力的维持方面发挥了重要作用。越来越多的证据表明，颗粒细胞的功能障碍会导致卵泡对促性腺激素的反应能力、卵母细胞发育等的异常，这些失调都与PCOS的发病机制密切相关。多个研究表明，在PCOS患者的颗粒细胞中发现了异常的基因表达谱，多个差异表达基因与PCOS的发病机制密切相关，然而这些基因的调控机制在很大程度上仍不清楚。申请人前期通过对从PCOS患者颗粒细胞中提取的RNA进行RNA-seq测序，对测序结果进行GO富集分析等生信分析后，我们发现，PCOS患者中m6A去甲基化酶ALKBH5的mRNA水平显著下降。
m6A是真核细胞中最常见的修饰形式。它在不改变碱基序列的条件下，能够调控RNA的转录、剪接、降解和翻译过程，并且这种修饰是动态的、可逆的，由三种不同类型的蛋白复合物调节，分别为m6A甲基转移酶(Writers)、m6A甲基化阅读蛋白(Readers)以及m6A去甲基酶(Erasers)。简而言之，m6A修饰通过m6A甲基转移酶和m6A去甲基酶来对RNA进行甲基化修饰或去甲基化，而m6A甲基化阅读蛋白则用来识别m6A修饰。随着高通量测序技术在检测m6A方面的应用，研究发现m6A修饰在终止密码子、3‘UTR区附近富集，并在5’UTR区附近以不依赖帽子结构的方式进行翻译。目前的研究表明，m6A甲基化水平与多种疾病的发生发展有关，例如肿瘤、阿尔茨海默病、牙周炎、心力衰竭、人腹主动脉瘤和肥胖症等。
ALKBH5作为一种重要的m6A去甲基化酶，在对RNA的m6A的修饰调控上发挥了重要作用。研究人员已经证明ALKBH5参与了细胞的增殖、迁移、侵袭、转移、肿瘤以及生殖系统疾病的发生发展。在生殖细胞中，ALKBH5作为主要的m6A修饰的“橡皮擦”，发挥着氧化逆转m6A的作用。研究人员发现，ALKBH5缺失的雄性小鼠不育，且其mRNA的m6A修饰增加，他们认为导致雄性小鼠不育的原因是由于m6A过度积累导致RNA的异常剪接，产生较短的转录本，导致精子减数分裂受阻。而ALKBH5在颗粒细胞中如何发挥作用，以及ALKBH5的缺失是否与多囊卵巢综合症的发生相关，目前仍不明确。
现有技术中，通过siRNA方法敲降ALKBH5基因，在研究中需要多次转染，这无疑增加了人力投入和研究成本，且会存在敲降效率不高及不稳定的现象。
因此，亟需一种ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株的构建方法。
发明内容
本发明的目的是针对现有技术的不足，提供一种ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株及其构建方法和应用，该细胞株可为研究ALKBH5对多囊卵巢综合症的致病机制的进一步深入研究搭建一个良好的基础。
本发明的目的是通过以下技术方案来实现的：本发明实施例的第一方面提供了一种ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株的构建方法，包括以下步骤：
(1)设计一对互补的靶向小鼠ALKBH5基因第一外显子的sgRNA，以PX459 V2.0质粒为载体，构建出一个重组质粒，所述sgRNA的序列包括如SEQ ID NO.1所示的正向引物ALKBH5-sgRNA F序列，和如SEQ ID NO.2所示的反向引物ALKBH5-sgRNA R序列；
(2)将所述重组质粒转染进经永生化处理的小鼠卵巢颗粒细胞中，使用嘌呤霉素进行阳性细胞筛选，分离单克隆细胞进行培养，获得ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株。
进一步地，所述步骤1中，所述以PX459 V2.0质粒为载体，构建出一个重组质粒包括如下子步骤：
(1.1)将两条所述sgRNA退火形成双链，经T4激酶磷酸化；
(1.2)使用Bbs I限制性核酸内切酶切割位于PX459 V2.0质粒第245和267个碱基上的Bbs I酶切位点，将质粒切割成线性化；然后利用SanPrep柱式PCR产物纯化试剂盒纯化回收DNA片段；
(1.3)将步骤(1.1)的产物与与步骤(1.2)回收的DNA片段利用T4 DNA连接酶进行连接，并转化至DH5α感受态细胞中，接种至带有氨苄抗性的平板上，37℃培养12-16h后，挑取单克隆菌落；
(1.4)用U6启动子序列通用引物进行测序，提取测序正确的质粒。
进一步地，所述步骤(1.1)中，两条sgRNA退火形成双链的过程具体为：将样品在37℃条件下孵育30min后，在95℃条件下处理2min，随后从50℃开始，以每秒下降0.1℃的速度进行退火，直到体系温度降至20℃，最后将样品放至4℃保存。
进一步地，所述步骤(2)具体包括以下子步骤：
(2.1)将经永生化处理的小鼠卵巢颗粒细胞接种到12孔板培养12-16小时，再将转染试剂和重组质粒按3uL：1ug的比例混匀后的转染复合物滴入孔中，轻摇细胞培养板使其分布均匀后，放入培养箱培养48小时；
(2.2)往细胞培养板中加入含5ug/mL嘌呤霉素的培养基，培养24h后更换含有同样浓度嘌呤霉素的培养基继续培养，后每48h更换一次含5ug/mL嘌呤霉素的新鲜培养基，直到转染组细胞长满培养皿并且几乎没有死亡脱落的细胞。
(2.3)将细胞消化制备细胞悬液，倍比稀释后接种至96孔板，使各孔含有0.5-1个细胞。隔日细胞贴壁后，观察单克隆的个数，并标记。待单克隆密度长到孔的50-60％时，胰酶消化细胞，获得ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株。
本发明实施例的第二方面提供了一种由上述的构建方法构建得到的ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株。
本发明实施例的第三方面提供了一种上述的ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株作为ALKBH5对多囊卵巢综合症的致病机制研究的细胞模型的应用。
本发明具有如下有益效果：本发明利用PX459 V2.0质粒载体构建的PX459 V2.0-ALKBH5携带有CRISPR/Cas9系统的Cas核酸内切酶基因；CRISPR/Cas基因编辑技术成功永久性的敲除了mGC细胞ALKBH5基因，获得了ALKBH5敲除的稳定细胞株，为研究ALKBH5对多囊卵巢综合症的致病机制的进一步深入研究搭建一个良好的基础。
附图说明
图1为本发明构建的含有敲除基因使用的引导sgRNA的重组质粒模式图；
图2为本发明提供的ALKBH5敲除的稳定mGC细胞株Alkbh5蛋白表达检测结果图；
图3为本发明提供的ALKBH5敲除的稳定mGC细胞株细胞自噬相关蛋白表达检测结果图。
具体实施方式
下面结合附图，对本发明进行详细说明。在不冲突的情况下，下述的实施例及实施方式中的特征可以相互组合。
实施例1：载体构建
本发明方法利用CRISPR/Cas 9相关技术，经过sgRNA的设计、插入片段的合成及酶切处理，构建出用于敲除ALKBH5的重组质粒，具体包括以下子步骤：
1)ALKBH5基因的sgRNA设计
设计一对互补的靶向小鼠ALKBH5基因第一外显子的sgRNA的过程具体为：sgRNAoligo序列的设计应用https://benchling.com网站设计一对针对ALKBH5 cds区的外显子的sgRNA序列。设计方法如下：利用该网站提供的基因数据库，选择我们所需要的目的基因，将其导入，在网站提供的序列结果中，选择on-target得分高，off-target得分高的序列，在其两端加上酶切位点。在每条sgRNA序列F链的5‘端添加CACCAAG，R链的5’端添加AAAC，3‘端添加C,与PX459 V2.0质粒经Bbs I酶切后形成的黏性末端互补。如F链的5‘端第一个碱基是G，则添加碱基时末尾不需要补G，相应的R链3’端也不需要补C。以下将PX459 V2.0重组质粒称为PX459 V2.0-ΔALKBH5。
表1：sgRNA寡核苷酸序列
2)将两条所述sgRNA退火形成双链，经T4激酶磷酸化
将两条sgRNA寡聚核苷酸单链退火形成双链，经T4激酶磷酸化，退火反应体系包括浓度为100uM的ALKBH5-sgRNA F和ALKBH5-sgRNA R所示引物各1uL、10*NEB buffer2 1uL、T4-PNK0.5uL、ddH2O6.5uL，放置PCR仪中反应，反应程序为37℃反应30min，95℃反应2min，再以0.1℃/s的速率，降温至20℃，即获得sgRNA的双链DNA片段，放4℃冰箱保存，准备与酶切载体连接。
3)载体酶切并回收
利用Bbs I限制性核酸内切酶将PX459 V2.0质粒进行线性化，然后用SanPrep柱式PCR产物纯化试剂盒(上海生工生物)纯化酶切产物。
具体步骤为：取PX459 V2.0载体1ug，用Bbs1酶切，酶切体系为：PX459 V2.0 1ug、Bbs1 1uL、10*NEB cut smart buffer 5ul，加ddH2O补足体系到50uL。混匀后37℃水浴1小时。使用酶切产物回收试剂盒对酶切产物进行回收纯化。
4)sgRNADNA片段与PX459 V2.0载体的DNA连接
酶连体系：2uL 10*T4buffer、1uL T4酶、2uL载体、10uL sgRNADNA片段，加ddH2O补足体系到20uL；混匀后于37℃水浴反应4小时。
5)连接产物转化及菌落PCR鉴定，构建出一个重组质粒
取上述连接产物5uL加入到50uL DH5α感受态细胞中，先冰浴30min，42℃水浴45s后冰上放置2min，加入到500uL不含抗生素的培养基中37℃摇床震荡45-60min；将菌液涂到氨苄抗性的平板上，37℃培养箱培养12-16h；挑取单克隆菌落进行菌落扩增，用U6启动子序列通用引物进行测序，鉴定正确的重组质粒PX459 V2.0-ALKBH5-sgRNA(如图1所示)进行菌液抽提重组质粒并保存。
实施例2：制备ALKBH5基因敲除小鼠卵巢颗粒细胞株
1)实验分组
分为阳性组(PX459 V2.0-ALKBH5-sgRNA质粒+转染试剂)，阴性对照组(阴性对照质粒+转染试剂)，空白对照组(转染试剂)；
2)细胞转染
本发明选用的小鼠卵巢颗粒细胞是由本实验室通过提取原代小鼠卵巢颗粒细胞后，将带有SV70基因片段的质粒载体转染进原代小鼠卵巢颗粒细胞，通过嘌呤霉素药物筛选的方法，得到抗性单克隆，扩大培养后建成小鼠卵巢颗粒细胞。随后，将mGC细胞以1.5*105/孔的密度接种到12孔板中，每孔含完全培养基1mL，每组设3组重复，放置细胞培养箱培养12-16小时，将孔内培养基更换为新鲜完全血清培养基。将37.5uL转染试剂lipofectamine3000(ThermoFisher Scientific)与12.5ug质粒分别逐滴加入到5mL不含血清的培养基的灭菌离心管中，混匀后室温孵育5min，再将混匀的转染试剂分别与PX459V2.0-ALKBH5-sgRNA质粒以及阴性对照质粒混合，室温孵育20min，每次取1mL转染复合物逐一滴入孔中。前后轻摇细胞培养板，使转染复合物在培养基中分布均匀。继续放入培养箱中培养，6小时更换新鲜完全培养基。
3)嘌呤霉素筛选
在培养48h后，往实验组和对照组细胞中分别加入含有终浓度5ug/mL嘌呤霉素的新鲜培养基；培养24h后更换含有同样浓度嘌呤霉素的新鲜完全培养基继续培养，之后每48h更换一次含5ug/mL嘌呤霉素的新鲜培养基，直到未转染组的细胞完全死亡，转染组细胞长满培养皿并且几乎没有死亡脱落的细胞。
4)细胞单克隆的筛选
将细胞消化后制备细胞悬液，细胞计数后用含有5ug/mL嘌呤霉素的新鲜培养基倍比稀释细胞悬液。在96孔板中，每孔加入100uL细胞悬液，使各孔含有0.5-1个细胞。隔日细胞贴壁后，观察单克隆的个数，并标记。待单克隆密度长到孔的50-60％时，用胰酶消化细胞，取50％细胞提取细胞基因组进行测序验证，剩下50％细胞继续在96孔板中培养扩增。
5)Western Blot鉴定Alkbh5蛋白表达量的降低情况
使用细胞裂解液裂解稳定敲除细胞株后，Pierce Rapid Gold BCA试剂盒测定蛋白样品浓度。蛋白样品经SDS-PAGE分离后转移到PVDF膜上，用含有5％脱脂奶粉的TBS将PVDF膜在室温下封闭1h，然后在4℃下与相应的一抗孵育过夜。次日，用TBST清洗膜1h，然后在相应的以辣根过氧化物酶结合的二抗中孵育30min。同样，在二抗体孵育后用TBST洗膜1h。最后用增强型化学发光底物和X射线胶片检测免疫反应条带。使用Image-Pro Plus软件对条带的强度进行量化。
如图2所示，通过免疫印迹分析，我们发现ΔALKBH5-mGC的Alkbh5蛋白与对照组相比明显降低，以此确定了ΔALKBH5-mGC细胞株中的Alkbh5蛋白被成功敲除。
实施例3：ALKBH5基因稳定敲除小鼠卵巢颗粒细胞株的应用
1)Real time RT-qPCR
①利用TRIzol试剂(Takara，日本)按说明提取总RNA后，用PrimeScript RT试剂盒(Takara)将3μg RNA逆转录成互补DNA(CDNA)。
②20μL反应体系
实时定量PCR 20μL反应体系如下：
③基因定量
以GAPD(3-磷酸甘油醛脱氢酶)作为内参，每个cDNA样本的重复3次，取平均值。目的基因mRNA的相对浓度用2-ΔDelta；Ct公式进行计算。
通过RT-qPCR分析，我们发现ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株中，与PCOS相关的基因Ptx3、Star的mRNA水平较对照组显著降低，而Has2的mRNA水平较对照组显著升高，同时，我们还发现自噬相关基因Bnip3的mRNA水平在ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株中显著升高。
2)Western Blot鉴定自噬相关蛋白表达量的变化情况
具体实验步骤同实施例2中的5)Western Blot鉴定Alkbh5蛋白表达量的降低情况。
由于有研究发现，自噬参与了PCOS相关的代谢紊乱，并且我们RT-qPCR的结果发现自噬相关基因Bnip3表达量在ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株中显著升高，因此，我们推测，Alkbh5表达的降低可能是通过自噬来影响PCOS的发生。
如图3所示，我们发现ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株中，自噬相关蛋白LC3-I/II、Pink1较对照组显著升高。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明保护的范围之内。
序列表
<110> 浙江大学医学院附属妇产科医院
<120> 一种ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株及构建方法和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
caccgcacca agcggaaata ccagg 25
<210> 2
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
aaaccctggt atttccgctt ggtgc 25
Claims (6)
1.一种ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株的构建方法，其特征在于，包括以下步骤：
(1)设计一对互补的靶向小鼠ALKBH5基因第一外显子的sgRNA，以PX459 V2.0质粒为载体，构建出一个重组质粒，所述sgRNA的序列包括如SEQ ID NO.1所示的正向引物ALKBH5-sgRNA F序列，和如SEQ ID NO.2所示的反向引物ALKBH5-sgRNA R序列；
(2)将所述重组质粒转染进经永生化处理的小鼠卵巢颗粒细胞中，使用嘌呤霉素进行阳性细胞筛选，分离单克隆细胞进行培养，获得ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株。
2.根据权利要求1所述的构建方法，其特征在于，所述步骤1中，所述以PX459 V2.0质粒为载体，构建出一个重组质粒包括如下子步骤：
(1.1)将两条所述sgRNA退火形成双链，经T4激酶磷酸化；
(1.2)使用Bbs I限制性核酸内切酶切割位于PX459 V2.0质粒第245和267个碱基上的Bbs I酶切位点，将质粒切割成线性化；然后利用SanPrep柱式PCR产物纯化试剂盒纯化回收DNA片段；
(1.3)将步骤(1.1)的产物与与步骤(1.2)回收的DNA片段利用T4 DNA连接酶进行连接，并转化至DH5α感受态细胞中，接种至带有氨苄抗性的平板上，37℃培养12-16h后，挑取单克隆菌落；
(1.4)用U6启动子序列通用引物进行测序，提取测序正确的质粒。
3.根据权利要求2所述的构建方法，其特征在于，所述步骤(1.1)中，两条sgRNA退火形成双链的过程具体为：将样品在37℃条件下孵育30min后，在95℃条件下处理2min，随后从50℃开始，以每秒下降0.1℃的速度进行退火，直到体系温度降至20℃，最后将样品放至4℃保存。
4.根据权利要求1所述的构建方法，其特征在于，所述步骤(2)具体包括以下子步骤：
(2.1)将经永生化处理的小鼠卵巢颗粒细胞接种到12孔板培养12-16小时，再将转染试剂和重组质粒按3uL：1ug的比例混匀后的转染复合物滴入孔中，轻摇细胞培养板使其分布均匀后，放入培养箱培养48小时；
(2.2)往细胞培养板中加入含5ug/mL嘌呤霉素的培养基，培养24h后更换含有同样浓度嘌呤霉素的培养基继续培养，后每48h更换一次含5ug/mL嘌呤霉素的新鲜培养基，直到转染组细胞长满培养皿并且几乎没有死亡脱落的细胞；
(2.3)将细胞消化制备细胞悬液，倍比稀释后接种至96孔板，使各孔含有0.5-1个细胞，隔日细胞贴壁后，观察单克隆的个数，并标记，待单克隆密度长到孔的50-60％时，胰酶消化细胞，获得ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株。
5.一种权利要求1-4中任一权利要求所述的构建方法构建得到的ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株。
6.一种权利要求5所述的ALKBH5基因稳定敲除的小鼠卵巢颗粒细胞株作为ALKBH5对多囊卵巢综合症的致病机制研究的细胞模型的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210729919.9A CN114934068B (zh) | 2022-06-24 | 2022-06-24 | Alkbh5基因稳定敲除的小鼠卵巢颗粒细胞株及构建方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210729919.9A CN114934068B (zh) | 2022-06-24 | 2022-06-24 | Alkbh5基因稳定敲除的小鼠卵巢颗粒细胞株及构建方法和应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114934068A CN114934068A (zh) | 2022-08-23 |
| CN114934068B true CN114934068B (zh) | 2024-04-19 |
Family
ID=82868175
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210729919.9A Active CN114934068B (zh) | 2022-06-24 | 2022-06-24 | Alkbh5基因稳定敲除的小鼠卵巢颗粒细胞株及构建方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114934068B (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108467864A (zh) * | 2018-04-04 | 2018-08-31 | 福州大学 | 一种icam-1基因敲除肿瘤细胞株及其应用 |
| CN112501170A (zh) * | 2020-11-30 | 2021-03-16 | 武汉爱博泰克生物科技有限公司 | 一种构建mlh1基因敲除细胞系的方法 |
| WO2021243881A1 (zh) * | 2020-06-01 | 2021-12-09 | 五邑大学 | 用于猪MBP基因敲除的sgRNA组合物及用途 |
-
2022
- 2022-06-24 CN CN202210729919.9A patent/CN114934068B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108467864A (zh) * | 2018-04-04 | 2018-08-31 | 福州大学 | 一种icam-1基因敲除肿瘤细胞株及其应用 |
| WO2021243881A1 (zh) * | 2020-06-01 | 2021-12-09 | 五邑大学 | 用于猪MBP基因敲除的sgRNA组合物及用途 |
| CN112501170A (zh) * | 2020-11-30 | 2021-03-16 | 武汉爱博泰克生物科技有限公司 | 一种构建mlh1基因敲除细胞系的方法 |
Non-Patent Citations (2)
| Title |
|---|
| ALKBH5 regulates anti-PD-1 therapy response by modulating lactate and suppressive immune cell accumulation in tumor microenvironment;Na Li等;PNAS;第117卷(第33期);第20161页左栏第一段，图1A，Supplementary Information第22页第二段，Datasets S4 * |
| m6A去甲基化酶ALKBH5对小鼠精原细胞功能的影响;夏维 等;中国生育健康杂志;第31卷(第5期);第488-490页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114934068A (zh) | 2022-08-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108410911B (zh) | 基于CRISPR/Cas9技术构建的LMNA基因敲除的细胞系 | |
| CN107502608A (zh) | 用于敲除人ALDH2基因的sgRNA、ALDH2基因缺失细胞株的构建方法及应用 | |
| CN110628766B (zh) | 与羊骨骼肌发育相关的lncRNA编码基因及其应用 | |
| CN110205327A (zh) | 一种水稻温敏核不育基因tms3突变体及其分子标记与应用 | |
| JPWO2003040363A1 (ja) | イネのトランスポゾン遺伝子 | |
| CN114934068B (zh) | Alkbh5基因稳定敲除的小鼠卵巢颗粒细胞株及构建方法和应用 | |
| CN110747230B (zh) | 一种促进牛骨骼肌卫星细胞成肌分化的方法 | |
| CN110862988A (zh) | 一种sgRNA及其构建的CREBRF点图变型巴马香猪和应用 | |
| CN115976021A (zh) | lncRNA MSTRG.5970.28及其应用、调控卵巢发育的产品和方法 | |
| CN112899279B (zh) | 一种构建Fzd6基因敲除小鼠模型的方法及应用 | |
| CN111073900B (zh) | 一种提高猪克隆胚胎发育效率的方法 | |
| CN114107176A (zh) | 一种稳定表达非洲猪瘟CD2v蛋白的CHO细胞系及其构建方法和应用 | |
| CN114032240A (zh) | 一种用于提高基因敲除效率的方法 | |
| CN107723293A (zh) | 一种棉纤维发育相关基因GbWRKY32及其表达载体与应用 | |
| CN111793624A (zh) | 一种定点敲除水稻OsAurora1基因的sgRNA的oligo DNA组 | |
| CN116286972A (zh) | 一种eif2b5基因敲除细胞系及其构建方法与应用 | |
| CN110129322A (zh) | Bna-miR393在调控甘蓝型油菜生殖器官发育过程中的应用 | |
| CN117660279B (zh) | 一种通过疮痂病链霉菌SCAB_75421基因提高thaxtomin A产量的方法 | |
| CN109652421B (zh) | 靶向编辑羊繁殖负调控基因NPFFR1的sgRNA及其编码DNA和应用 | |
| LU102620B1 (en) | A gRNA and Related Kit and Application | |
| WO2024098353A1 (zh) | 一种产大麻萜酚酸的重组酿酒酵母及其构建方法和应用 | |
| CN111793653B (zh) | 一种dpy19l1l基因缺失型斑马鱼的构建方法 | |
| CN114891786B (zh) | 犬Rosa26基因及其应用 | |
| CN115806939A (zh) | 一种Apaf1基因敲除HEK293细胞系及构建方法和应用 | |
| CN116376965A (zh) | GhPIP5K9a基因在调控植物主根发育中的应用 |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |