CN116334079A - 靶向Was基因的双sgRNA和敲除Was基因的mESC细胞系及其应用 - Google Patents
靶向Was基因的双sgRNA和敲除Was基因的mESC细胞系及其应用 Download PDFInfo
- Publication number
- CN116334079A CN116334079A CN202211040899.0A CN202211040899A CN116334079A CN 116334079 A CN116334079 A CN 116334079A CN 202211040899 A CN202211040899 A CN 202211040899A CN 116334079 A CN116334079 A CN 116334079A
- Authority
- CN
- China
- Prior art keywords
- gene
- cell line
- targeting
- mesc
- cell
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 49
- 108091027544 Subgenomic mRNA Proteins 0.000 title claims abstract description 23
- 230000008685 targeting Effects 0.000 title claims abstract description 17
- 238000003209 gene knockout Methods 0.000 claims abstract description 7
- 230000009977 dual effect Effects 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 80
- 108091033409 CRISPR Proteins 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 13
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 11
- 238000010354 CRISPR gene editing Methods 0.000 claims description 7
- 239000013598 vector Substances 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 5
- 239000012620 biological material Substances 0.000 claims description 4
- 238000011160 research Methods 0.000 claims description 3
- 108020004414 DNA Proteins 0.000 claims description 2
- 230000009261 transgenic effect Effects 0.000 claims description 2
- 102000053602 DNA Human genes 0.000 claims 1
- 239000012634 fragment Substances 0.000 abstract description 9
- 208000011580 syndromic disease Diseases 0.000 abstract description 9
- 238000012216 screening Methods 0.000 abstract description 8
- 230000000694 effects Effects 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 19
- 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 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 15
- 238000004458 analytical method Methods 0.000 description 14
- 238000001514 detection method Methods 0.000 description 10
- 239000002609 medium Substances 0.000 description 10
- 238000012163 sequencing technique Methods 0.000 description 10
- 238000004043 dyeing Methods 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 241000204031 Mycoplasma Species 0.000 description 8
- 229950010131 puromycin Drugs 0.000 description 8
- 238000007480 sanger sequencing Methods 0.000 description 8
- 238000010186 staining Methods 0.000 description 8
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 241000282326 Felis catus Species 0.000 description 6
- 210000000130 stem cell Anatomy 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 241000699670 Mus sp. Species 0.000 description 5
- 206010028980 Neoplasm Diseases 0.000 description 5
- 239000006285 cell suspension Substances 0.000 description 5
- 238000010166 immunofluorescence Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 239000013641 positive control Substances 0.000 description 5
- 238000002791 soaking Methods 0.000 description 5
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 4
- 241001529936 Murinae Species 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 239000012295 chemical reaction liquid Substances 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 238000010362 genome editing Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 229910052754 neon Inorganic materials 0.000 description 4
- GKAOGPIIYCISHV-UHFFFAOYSA-N neon atom Chemical compound [Ne] GKAOGPIIYCISHV-UHFFFAOYSA-N 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 206010043276 Teratoma Diseases 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 210000000349 chromosome Anatomy 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 2
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 101150037203 Sox2 gene Proteins 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 210000001654 germ layer Anatomy 0.000 description 2
- 108010082117 matrigel Proteins 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000009437 off-target effect Effects 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 206010043554 thrombocytopenia Diseases 0.000 description 2
- 230000005740 tumor formation Effects 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 239000005660 Abamectin Substances 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 108020005004 Guide RNA Proteins 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 244000179970 Monarda didyma Species 0.000 description 1
- 235000010672 Monarda didyma Nutrition 0.000 description 1
- 206010028470 Mycoplasma infections Diseases 0.000 description 1
- 241000204003 Mycoplasmatales Species 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 208000013544 Platelet disease Diseases 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 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 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- RRZXIRBKKLTSOM-XPNPUAGNSA-N avermectin B1a Chemical compound C1=C[C@H](C)[C@@H]([C@@H](C)CC)O[C@]11O[C@H](C\C=C(C)\[C@@H](O[C@@H]2O[C@@H](C)[C@H](O[C@@H]3O[C@@H](C)[C@H](O)[C@@H](OC)C3)[C@@H](OC)C2)[C@@H](C)\C=C\C=C/2[C@]3([C@H](C(=O)O4)C=C(C)[C@@H](O)[C@H]3OC\2)O)C[C@H]4C1 RRZXIRBKKLTSOM-XPNPUAGNSA-N 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000002354 daily effect Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 231100000221 frame shift mutation induction Toxicity 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000033065 inborn errors of immunity Diseases 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000010339 medical test Methods 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 235000012736 patent blue V Nutrition 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000003950 pathogenic mechanism Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-N picric acid Chemical compound OC1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-N 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 208000028529 primary immunodeficiency disease Diseases 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 238000011476 stem cell transplantation Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C12N15/11—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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- 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
- 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
- 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
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
-
- 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 RNAses, DNAses
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
- A01K2267/0325—Animal model for autoimmune diseases
-
- 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
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/91—Cell lines ; Processes using cell lines
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Developmental Biology & Embryology (AREA)
- Gynecology & Obstetrics (AREA)
- Medicinal Chemistry (AREA)
- Environmental Sciences (AREA)
- Physics & Mathematics (AREA)
- Reproductive Health (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Toxicology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明属于基因工程技术领域,具体涉及靶向Was基因的双sgRNA和敲除Was基因的mESC细胞系及其应用。所述靶向Was基因的双sgRNA,包括Was‑sgRNA1和Was‑sgRNA2;所述Was‑sgRNA1的靶向序列为:CACAGTTGTTCAGCTCTACC;所述Was‑sgRNA2的靶向序列为:GGATGAAGTAGGACTTCTGA。本发明采用两条分别靶向小鼠Was基因第2外显子的sgRNA序列,可通过一次细胞筛选直接获得大片段基因敲除细胞系,敲除效果更加彻底,能够更好地模拟WAS综合征的重症表型。
Description
技术领域
本发明属于基因工程技术领域,具体涉及靶向Was基因的双sgRNA和敲除Was基因的 mESC细胞系及其应用。
背景技术
湿疹血小板减少伴免疫缺陷综合征(Wiskott-Aldrich Syndrome,WAS)是一种少见的X 连锁隐性遗传病,以湿疹、血小板减少和免疫缺陷为临床表现,易患自身免疫病和恶性肿瘤。 新生儿WAS发病率为(1~10)/百万,多发生于男性。此外,WAS患者的血小板异常以及自 身免疫疾病方面的成因也不清楚。WAS综合征的临床症状非常复杂,有待于建立高效系统的 诊断方法,其主要治疗手段为干细胞移植重建免疫功能。为了更好地理解WAS综合征的致病 机理,发展高效准确的诊断方法,提供有效的治疗方案,本发明构建了Was缺失的小鼠胚胎 干细胞(mESC)系。
发明内容
本发明旨在提供靶向Was基因的双sgRNA和敲除Was基因的mESC细胞系及其应用。
为了达到上述目的,本发明采用以下技术方案:靶向Was基因的双sgRNA,包括Was-sgRNA1和Was-sgRNA2;
所述Was-sgRNA1的靶向序列为:CACAGTTGTTCAGCTCTACC;
所述Was-sgRNA2的靶向序列为:GGATGAAGTAGGACTTCTGA。
优选地,所述Was-sgRNA1的核苷酸序列包括:Was-sgRNA1 Oligo: 5’-tatcttgtggaaaggacgaaacaccCACAGTTGTTCAGCTCTACCgttttagagctagaaatagca-3’;
所述Was-sgRNA2的核苷酸序列包括:Was-sgRNA2 Oligo: 5’-gctatttctagctctaaaacTCAGAAGTCCTACTTCATCCaggtcccaccgagatttgaac-3’。
本发明提供用于编码所述靶向Was基因的双sgRNA的DNA分子。
本发明提供包括所述靶向Was基因的双sgRNA的生物材料,所述生物材料为载体、表达 盒或转基因细胞中的一种或多种。
本发明还提供一种敲除了Was基因的细胞系的构建方法,包括:利用CRISPR/Cas9系统 敲除所述细胞系的Was基因,所述CRISPR/Cas9系统使用的sgRNA为所述靶向Was基因的 双sgRNA。
优选地,所述利用CRISPR/Cas9系统敲除所述细胞系的Was基因中使用pU6-gRNA载体。
优选地,所述细胞系为小鼠胚胎干细胞系。
本发明还提供一种小鼠胚胎干细胞系,所述小鼠胚胎干细胞系由所述方法构建得到。
本发明还提供所述小鼠胚胎干细胞系在构建Was基因功能研究模型中的应用。
WAS综合征存在多种基因突变,但是WAS综合征的临床表现与其编码的WAS蛋白的表 达量成反比,WAS蛋白的表达量越低,WAS综合征的临床表现越严重,为了更好的模拟WAS综合征的重症表型,本发明采用了两条sgRNA对小鼠胚胎干细胞的Was基因进行删除,增加基因组片段缺失的频率,较于单位点敲除法敲除效率高、种系突变多,更易造成移码突变,并 且由于本发明的2个sgRNA设计在同一个外显子上,即使没有片段丢失,2个sgRNA造成阅读框发生错位突变的概率也远大于单独一个sgRNA。
与现有技术相比,本发明具有以下有益效果:
(1)本发明采用两条分别靶向小鼠Was基因第2外显子的sgRNA序列,可通过一次细胞筛选直接获得大片段基因敲除细胞系,发明中基于CRISPR/Cas9系统敲除Was基因的方法, 与沉默、敲低、干扰等方法相比,敲除效果更加彻底,可以更好地模拟WAS综合征的重症表 型。
(2)通过本发明获得的小鼠Was基因敲除细胞系可直接用于体细胞克隆获得基因修饰鼠, 进而在今后相关疾病的致病机制及疾病治疗方案研究方面发挥更大的作用。
附图说明
图1为本发明针对鼠Was基因第2外显子的sgRNA1序列与sgRNA2序列示意图。
图2为本发明pU6-gRNA的基因图谱。
图3为本发明pU6-gRNA酶切结果图。
图4为本发明质粒PCR跑胶结果图。
图5为本发明sgRNA1,sgRNA2测序结果图。
图6为本发明混合细胞sanger测序结果图。
图7为本发明21号mESC sanger测序图。
图8为本发明21号mESC T载体连接测序结果图。
图9为本发明21号mESC核型分析图。
图10为本发明21号mESC支原体检测结果图。
图11为本发明脱靶位点的sanger测序结果图。
图12为本发明外源基因整合检测图。
图13为本发明21号mESC细胞免疫荧光图。
图14为本发明小鼠背部两侧的畸胎瘤图。
图15为本发明21号畸胎瘤切片后HE染色结果图。
图16为本发明21号mESC碱性磷酸酶染色结果图。
具体实施方式
下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然, 所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例, 本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保 护的范围。
实施例中,所使用的实验方法如无特殊说明,均为常规方法,所用的材料、试剂等,如 无特殊说明,均可从商业途径得到。
实施例1、CRISPR Cas9质粒构建
1.1Cas9靶位点的选择
Cas9靶位点包含20个碱基,紧邻靶点3’端的3个碱基构成PAM区,PAM区要求序列为NGG(N为任意碱基)。参考如下的Cas9靶位点预测网站:(http://crispr.mit.edu),根据该原 则设计针对鼠Was基因第2外显子的双sgRNA序列(鼠Was基因在ncbi中的Gene ID:22376), 两条靶序列分别为Was-sgRNA1:CACAGTTGTTCAGCTCTACC(如SEQ ID NO:1所示),Was-sgRNA2:GGATGAAGTAGGACTTCTGA(如SEQ ID NO:2所示)。
1.2根据确定好的靶点合成相应的oligos
将两条针对鼠Was基因第2外显子的Was-sgRNA1,Was-sgRNA2序列共同连接在pU6-gRNA中,两条oligo分别如表1所示,oligo中大写部分为sgRNA序列。
表1oligo序列
1.3pU6-gRNA质粒载体构建过程
(1)BbsI限制性内切酶(NEB,R3539S)酶切骨架质粒pU6-gRNA,酶切体系如下表2,37℃酶切3h左右,回收之前先取5uL产物跑胶确认酶切完全;
表2酶切体系
(2)采用胶回收试剂盒(TIANGEN)回收酶切产物(3950bp),参见附图3;
(3)以pU6-gtRNA-gag为模板,oligo为引物进行PCR扩增,回收241bp的片段。使用2×phanta Max Master mix(Vazyme)按下表3配制体系后,进行PCR。PCR条件如表4所示,质粒PCR跑胶结果如附图4所示。
表3PCR扩增体系
表4PCR反应条件
(4)使用重组酶Exnase Multis(Vazyme)进行重组,反应体系见表5,在37℃重组30min;
表5重组体系
(5)将重组后的体系转化大肠杆菌DH5α感受态(康体生命cat:KTSM101L),加入LB培养基,37℃、180rpm/min培养2h,取100μl菌液涂布于固体LB培养基(氨苄100μg/ml), 37℃倒置过夜培养,挑菌,测序(M13F,测序公司通用引物),质粒测序正确如附图5所示, 图中标记的部分为sgRNA序列;
(6)选取正确的质粒用无内毒素小提中量试剂盒(TIANGEN)提取质粒,-20℃冷冻备 用。
实施例2、小鼠胚胎干细胞Was基因敲除细胞的筛选
本实施例2中所使用的mESC培养基均为DME/F-12 1:1mixture(SIGMA,cat:D6421-500ml), 具体培养基组分如表6所示。
2.1嘌呤霉素筛选浓度的确定
(1)小鼠胚胎干细胞(赛业生物科技,MUAES-01001)生长至80%后以1:8传代;
(2)24h后,加入含不同浓度嘌呤霉素的新鲜mESC培养基(嘌呤霉素的浓度梯度分别 为:0.5ug/ml,1ug/ml,1.25ug/ml,1.5ug/ml,2ug/ml);
(3)每日显微镜下观察细胞存活比例,嘌呤霉素的最佳作用时间一般在1-4天之间;
(4)选用加药2-3天杀死所有细胞的最低筛选浓度作为筛选细胞时选用的嘌呤霉素浓度, 本实验的嘌呤霉素筛选浓度为1ug/mL。
2.2细胞的转染与筛选
2.2.1提前复苏mESC至6孔板中,培养细胞准备转染,细胞转染步骤如下:
1)收集细胞密度在80%的mESC;
2)吸弃培养基,用PBS洗两遍,加入0.05%Trypsin-EDTA(gibco),37℃孵育3分钟;
3)加入1mLmESC培养基(DME/F-12,1:1mixture(SIGMA,cat:D6421-500ml),其中各种 成分如表6所示)终止消化,吹打混匀后转移至15mL离心管中,同时取10ul细胞进行细胞计数,计算得到的细胞个数为3.8×106;
4)将离心管的细胞离心:在1000rpm,25℃离心5分钟;
5)吸弃上清,注意不要碰到细胞沉淀,加入760ul PBS重悬细胞,取90ul的细胞到1.5ml 管中,1000rpm,25℃离心5分钟,弃掉上清;
6)加入90ul电转专用重悬缓冲液Resuspension buffer(invitrogen Neon),充分重悬细胞;
7)使用转染系统(invitrogen Neon)进行电转,将电转专用导电液Electrolyticbuffer加入到 电击杯中,设置好相应条件(如表7所示);
8)将4.5ul实施例1步骤1.3制备的pU6-gRNA(1088ng/μl)与4.5ul px459(柯雷生物, kl-zl-0297,1007ng/μl)加入到90ul悬浮细胞中,混匀(不能产生气泡),随后用10ulNeon电 击枪吸取10uL的悬浮细胞,插入电击杯中;
9)按下Neon控制装置上的开始(start)按钮,完成后将电击枪取下来,吸出细胞打入(悬 空,吸管勿接触液面)预先铺上gelatin(STEMCELL,07903)的6孔板中,每孔添加有2ml含 mLif(Merck Millipore,ESG1107)的mESC培养基,随后用PBS润洗电击枪;
10)将培养板置于37℃和5%CO2的培养箱中;
11)由于刚电转的细胞活力较弱,所以培养三天后再使用嘌呤霉素进行筛选,以备挑选单 克隆。
表6 50ml mESC培养基的组分及体积用量
表7mESC电转染条件
2.2.2单克隆挑选:
1)三天后加入1μg/mL的嘌呤霉素筛选阳性细胞,筛选后的细胞长到75%的汇合度,加 入0.05%Trypsin-EDTA(gibco),37℃孵育3分钟;
2)加入1mL mESC培养基终止消化,吹打混匀后转移至15mL离心管中;
3)将离心管的细胞离心:在1000rpm,25℃离心5分钟,吸弃上清,注意不要碰到细胞 沉淀;
4)用1ml含mlif的mESC培养基重悬细胞,以每孔500个传代于三个10cm皿(需提前包被gelatin)中,剩余细胞收集起来,进行细胞裂解后PCR,Sanger测序看靶位点有无基因编辑;
5)分板24h后,用含mlif的mESC培养基换液,以后根据细胞培养情况一般两天换一次 液,每天观察细胞;
6)细胞培养10-14天左右在显微镜下可看到细胞克隆,用marker笔画出显微镜下观察到 的细胞克隆,挑选克隆前,首先用gelatn包被48孔板,每孔添加0.25ml mESC培养基,随后 在超净工作台显微镜下,使用10μl移液枪进行克隆刮取,将每个细胞克隆挑取至48孔板中, 待48孔细胞克隆长至80%时,传代至24孔,并取出部分细胞进行提取基因组后PCR,测序 鉴定。鉴定所用的上游引物一般设计在sgRNA位点上游100-200bp处,下游引物一般设计在 sgRNA位点下游100-200bp处;
7)细胞基因组提取:将部分鼠胚胎干细胞处理为细胞悬液,以10000rpm离心1分钟, 弃尽上清,加入30μl NP40裂解液振荡混匀后放入PCR仪中,PCR反应条件:56℃1h,95℃10min。
2.2.3小鼠胚胎干细胞sanger测序结果:
本次实验的鉴定oligo为鉴定-F1与鉴定-R1,具体见表8,鉴定PCR扩增条件见表9,PCR 条带的大小为417bp。
表8鉴定oligo序列
表9PCR反应条件
1)为确定混合细胞基因靶点有无发生编辑,若发生了基因编辑,则可以进一步挑取单克 隆。收集混合细胞PCR,随后进行sanger测序,结果如附图6所示,图A是靶位点的基因序 列,图B是mESC的野生型(WT)和电转染后的混合细胞(Was-ko)的sanger测序图。由附图 6可知,混合细胞发生了在靶点处的基因编辑;
2)挑取的细胞克隆打靶位点基因鉴定:挑取的细胞克隆中,获得大片段基因敲除纯合子 克隆有1个,为21号。Sanger测序如附图7,标记的部分是sgRNA1的序列。从附图7中可 以看出21号发生了靶位点的基因编辑。接着,对21号连接T载体(PMD19-T VectorTaKaRa) 分析后,测序分析结果如附图8所示,由测序结果可知,21号为通过基因编辑获得的大片段 基因敲除纯合子克隆;
3)鉴定后,将mES阳性克隆21号进行扩大培养,以进行下一步实验。
实施例3、Was基因缺失的mESC细胞系的安全性分析与多能性鉴定分析
Was基因大片段缺失的阳性克隆21号的安全性分析包括核型分析、支原体检测、外源基 因整合检测、脱靶检测:
3.1核型分析
mESC于T25细胞培养瓶(BIOFIL)中生长至汇合度80%时,换新鲜mESC培养基并将培养基加满T25细胞培养瓶,快递送到佛山迪安医学检验实验室有限公司做核型分析,结果显示mESC阳性克隆21号为正常核型,参考附图9。
通过细胞染色体核型分析,可以得出染色体数量以及结构变异的重要信息,辅助进行病理 分析和诊断干细胞研究。因此对干细胞进行染色体核型分析结果证明了干细胞的质量良好。
3.2支原体检测
支原体污染将使得用被污染的细胞样本实验完全失去意义和价值。本次实验支原体检测选 用的支原体检测试剂是MycoBlue-Mycoplasma Detector(Vazyme,D101)。
1)在实施例2制备的mESC阳性克隆21号换液三天,汇合度达到90%时取30μl培养液 上清置于PCR管中备用;
2)将MycoBlue Buffer从-20℃冰箱取出,待解冻后,颠倒混匀。根据待测样品数量在微 量离心管中配制如表10反应体系。用移液器轻轻吹打均匀,分装到PCR管中,每支反应管 25ul;
表10支原体检测反应体系
3)加样:
样品:向反应体系中加入1μl待测培养液上清;
阳性对照:向反应体系中加入1μl Positive Control;
阴性对照:25μl反应体系中不加入任何样品;
4)反应:将反应管放入PCR仪中,温度设置成60℃,孵育60min,反应结束后,在一个光线良好的环境中观察反应液颜色(以白纸为背景)。如果反应液仍为紫色,则判定为支原体 阴性;如果反应液变成天蓝色,则判定为支原体阳性。
检测结果表明样本mESC阳性克隆21号为阴性,说明没有支原体感染,结果见附图10 (1,2,3号分别是阳性对照,阴性对照,21号)。
3.3脱靶分析
脱靶效应会带来大量的非靶序列的单核苷酸变异(SNVs)和InDels,这些突变涉及了基因组 的编码区和非编码区,甚至有可能会直接影响机体的生理功能,因此需要评估脱靶效应造成的 影响。
脱靶分析:利用Cas-OFFinder分析得到gRNA位点的脱靶位点,脱靶位点信息如表11。 针对表11中的10个潜在的脱靶位点设计10对oligos序列,如表12所示。使用2×phantaMax Master mix(Vazyme)酶,配制10μl PCR体系,如表13所示,PCR体系的反应条件如表14所示。
将其中扩增片段大小符合预期的PCR产物送去Sanger测序,测序结果表明10个位点都 与野生型一样,并没有发生突变,说明没有发生脱靶,结果如附图11所示。
表11脱靶位点序列信息
表12脱靶位点oligo序列
oligo | oligo序列(5’-3’) | oligo | oligo序列(5’-3’) |
Primer1-F | CCAAGAGAGCAGGTTGGCAT | Primer1-R | GCTTAAAGACTGTTCATCACTGCT |
Primer2-F | AACAGGACTGGCACATGAAA | Primer2-R | GCAATCATGTGGAGTTGCAG |
Primer3-F | GTGACATCCAACCACCAACA | Primer3-R | GATCTGATGCCCTCTTCTGC |
Primer4-F | ATGCATTTGCACAGGGTACA | Primer4-R | CCCTCTGTCAGATGTGGAGT |
Primer5-F | GTTCTGGGAAAGCCAAACAA | Primer5-R | AGGATGAGCGTCTGCAAAGT |
Primer6-F | CAACATCCCCCTGCTTAAAA | Primer6-R | TTCACAGGAGTGCATGGAAG |
Primer7-F | GTAGACCCCGTGTCCGTCTA | Primer7-R | TGCCCCTAATGAGGATGAAG |
Primer8-F | GAGCTGAGGGTGCAAAGAAG | Primer8-R | TGTTTTGTTTCGGGCTTTTC |
Primer9-F | AGAGTGACTCCCCCAGAGGT | Primer9-R | CATGCTCATGGATTGTCAGG |
Primer10-F | AGGGAGTGGCATATGGAGTG | Primer10-R | GCTATGGTCATGGTGGCTCT |
表13PCR体系
表14PCR反应条件
3.4外源基因整合检测
针对外源表达载体PX459是否整合进mESC基因组,设计引物F1和R1序列如表15,PCR 条带大小为450bp,若外源基因整合进基因组,则可以看到mESC组出现450bp左右的条带。 使用2×phanta Max Master mix(Vazyme)酶,配制20μl PCR体系,如表16所示,PCR反应 条件如表17所示。
以水和Px459质粒分别作为阴性和阳性对照,对反应产物进行凝胶电泳鉴定,可看到阳性 对照有明显条带,而mES的WT与21号和阴性对照均没有条带,说明外源基因没有整合进基 因组。结果如附图12所示。
表15oligo序列
表16PCR体系
表17PCR反应条件
实施例4、Was基因缺失的mESC细胞系的多能性鉴定分析
4.1细胞免疫荧光实验
将实施例2制备的mESC阳性克隆21号传代至四孔板中的三个孔,待细胞密度为40%后, 开始进行免疫荧光实验。
1)将四孔板中的培养基吸出,用PBS浸洗3次,每次3min;
2)用4%的多聚甲醛(Roles-Bio,RBG1116-1)固定细胞15min,PBS浸洗3次,每次3min;
3)加入PBS配置的0.5%Trition X-100(Beyotime,ST795)室温通透20min;
4)PBS浸洗3次,每次3min,吸干PBS,滴加BSA封闭液(Solarbio,SW3015),室 温封闭30min;
5)吸干封闭液,每个孔直接加入200μl稀释好(用PBS稀释,稀释比例1:200)的一抗: 分别加入Oct4(abcam cat:ab181557),Sox2(abcam cat:ab137385),Nanog(abcam,cat:ab80892), 4℃孵育过夜;
第二天:
6)加荧光二抗:将一抗吸出,用PBST浸洗3次,每次3min;吸干PBS,加入用PBS 1:2000 稀释好的荧光二抗Goat Anti-Rabbit lgG H&L(Alexa488),用锡箔纸包裹室温孵育60 min;将二抗吸出,用PBST浸洗3次,每次3min(从加入二抗后,后面所有的步骤都要避光 进行);
7)每个孔都加入稀释好的DAPI(Beyotime,C1002)避光孵育5min,对细胞进行染核, 用PBS浸洗4次,每次5min,洗去多余的DAPI;每个孔换上新的PBS到荧光显微镜下观察拍照。
细胞的免疫荧光结果如附图13所示,细胞免疫荧光结果显示Oct4、Sox2和Nanog三个 多能性的Marker都正常表达,说明敲除Was基因的mESC是具有多能性的。
4.2三胚层分化鉴定
1)准备三个细胞量在80%的10cm培养皿的mESC阳性克隆21号,DPBS清洗细胞两次, 加入1.5ml 0.05%Trypsin-EDTA(gibco),37℃消化2min,细胞大部分脱落,加入4mLmESC 培养基终止消化,分别转移至三个15mL离心管,离心(1000rpm,5min);
2)吸去上清,DPBS清洗、重悬,再离心(1000rpm,5min);
3)用DMEM/F12(SIGMA,D6421)培养基重悬细胞,计数,使细胞终浓度达为 1×107cells/mL。
4)1.5ml EP管中加入300ul Matrigel(Corning,356255)后,再加入300ul细胞悬液,然 后加入0.6ul的Y-27632(MedChemExpress,HY10071),轻轻混合均匀(Matrigel胶比较黏, 注意不要吹出大量气泡),置于冰上;
5)月龄在1.5~2的免疫缺陷小鼠(约25g/只),腹腔注射阿佛汀麻醉,待小鼠身体变软 后,背部两侧予以剃毛处理;
6)细胞注射之前瞬时离心一下,然后用200ul枪头把细胞悬液混匀;再用1ml注射器去 掉针头后吸取悬液,每只小鼠每侧背部皮下注射250μL细胞悬液,每管细胞注射一只小鼠的 两个点,注射过程可观察到隆起;
7)注射完毕后,苦味酸标记细胞注射位置,方便后续取材;
8)每隔一个礼拜观察小鼠状态,2个礼拜后开始检测注射部位是否有肿瘤形成,确认有 肿瘤形成的话,取出肿瘤从中间切开后加固定液固定。肿瘤照片如附图14所示(图中肿瘤来 自两只小鼠的背部两侧的畸胎瘤);
9)肿瘤固定48小时后送至广州塞维尔生物科技有限公司进行石蜡包埋、切片和HE染色。
HE染色结果表明,肿瘤中有三个不同胚层组织的形成(参考附图15),说明Was基因敲 除的mESC阳性克隆21号具有体内分化成三个不同胚层的能力。
4.3碱性磷酸酶染色
使用懋康生物的碱性磷酸酶染色试剂盒(干细胞专用)(CAT:MP7503)对mESC阳性克 隆21号进行染色。
1)实验前准备材料:PBS缓冲液、PBST缓冲液(取5ul的Tween到10ml 1×PBS中,使其终浓度为0.05%,充分混匀即可);
2)染色工作液准备:按照如下比例溶液A:溶液B:溶液C=1:1:1配制适量染色工作液 (为了保证最佳染色结果,需在配制30min内用完染色工作液)。本次染色使用24孔板,每个孔配置0.6ml染色液;
3)染色步骤:吸出细胞培养液,PBST洗3次,用0.5ml固定液室温固定5min。【注意】:固定不要超过5min,过度固定会导致ALP失活。
4)吸出固定液,PBST洗2次,吸出PBST。加入0.6ml/孔染色工作液覆盖细胞,室温避光放置15min;【注意】:染色过程需密切监督颜色变化,当颜色很明亮即可终止反应,否则会引起非特异性染色;
5)吸出染色工作液,用PBS洗2次,最后保存于PBS,显微镜下观察染色结果。
结果如附图16显示,左侧图片为mESC阳性克隆21号在显微镜明场下的照片,右侧为 21号mESC碱性磷酸酶染色图片,细胞染色显示红色,判定细胞未发生分化。
上述实施例仅例示性说明本发明的原理及其功效,而非用于限制本发明。任何熟悉此技术 的人士皆可在不违背本发明的精神及范畴下,对上述实施例进行修饰或改变。因此,举凡所属 技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修 饰或改变,仍应由本发明的权利要求所涵盖。
Claims (9)
1.靶向Was基因的双sgRNA,其特征在于,包括Was-sgRNA1和Was-sgRNA2;
所述Was-sgRNA1的靶向序列为:CACAGTTGTTCAGCTCTACC;
所述Was-sgRNA2的靶向序列为:GGATGAAGTAGGACTTCTGA。
2.根据权利要求1所述靶向Was基因的双sgRNA,其特征在于,所述Was-sgRNA1的核苷酸序列包括:Was-sgRNA1 Oligo:
5’-tatcttgtggaaaggacgaaacaccCACAGTTGTTCAGCTCTACCgttttagagctagaaatagca-3’;
所述Was-sgRNA2的核苷酸序列包括:Was-sgRNA2 Oligo:
5’-gctatttctagctctaaaacTCAGAAGTCCTACTTCATCCaggtcccaccgagatttgaac-3’。
3.用于编码权利要求1或2所述靶向Was基因的双sgRNA的DNA分子。
4.一种生物材料,其特征在于,包含权利要求1或2所述靶向Was基因的双sgRNA,所述生物材料为载体、表达盒或转基因细胞中的一种或多种。
5.一种敲除了Was基因的细胞系的构建方法,其特征在于,包括:利用CRISPR/Cas9系统敲除所述细胞系的Was基因,所述CRISPR/Cas9系统使用的sgRNA为权利要求1或权利要求2所述的靶向Was基因的双sgRNA。
6.根据权利要求5所述的构建方法,其特征在于,所述利用CRISPR/Cas9系统敲除所述细胞系的Was基因中使用pU6-gRNA载体。
7.根据权利要求5所述的构建方法,其特征在于,所述细胞系为小鼠胚胎干细胞系。
8.一种小鼠胚胎干细胞系,其特征在于,所述小鼠胚胎干细胞系由权利要求5或权利要求6所述方法构建得到。
9.权利要求8所述小鼠胚胎干细胞系在构建Was基因功能研究模型中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211040899.0A CN116334079A (zh) | 2022-08-29 | 2022-08-29 | 靶向Was基因的双sgRNA和敲除Was基因的mESC细胞系及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211040899.0A CN116334079A (zh) | 2022-08-29 | 2022-08-29 | 靶向Was基因的双sgRNA和敲除Was基因的mESC细胞系及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116334079A true CN116334079A (zh) | 2023-06-27 |
Family
ID=86888151
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211040899.0A Pending CN116334079A (zh) | 2022-08-29 | 2022-08-29 | 靶向Was基因的双sgRNA和敲除Was基因的mESC细胞系及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116334079A (zh) |
-
2022
- 2022-08-29 CN CN202211040899.0A patent/CN116334079A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111172114A (zh) | 一种人源化肠癌前病变永生化上皮细胞系、构建方法及其应用 | |
CN113604504A (zh) | 用于pAPN基因16外显子定点修饰的组合物及其应用 | |
CN106755087A (zh) | 稳定表达猪瘟病毒e2蛋白的重组细胞系、制备方法、应用、及猪瘟病毒亚单位疫苗 | |
CN111454990B (zh) | 人颈静脉球副神经节瘤永生化细胞株及其应用 | |
CN116334079A (zh) | 靶向Was基因的双sgRNA和敲除Was基因的mESC细胞系及其应用 | |
CN104531607B (zh) | 犬原代细支气管上皮细胞及其在制备永生化细胞中的应用 | |
CN113046322B (zh) | 一种永生化的奶牛胎盘滋养层细胞系及其构建方法 | |
CN105063023A (zh) | 一种锌指核酸酶介导的猪mstn基因突变序列及其应用 | |
CN113166763B (zh) | 靶向cyp4v2基因突变位点的核酸分子及其用途 | |
CN114934066A (zh) | 石骨症的基因编辑体系及其应用 | |
CN110042123B (zh) | 一种通过诱导表达zfp57提高牛体细胞克隆效率的方法 | |
CN111269940A (zh) | 一种使用转录因子foxo1直接转分化间充质干细胞为精子的方法 | |
CN111254120A (zh) | 一种EpCAM基因人源化小鼠肿瘤细胞模型、构建方法以及用途 | |
CN117384856B (zh) | 一种永生化copd人支气管上皮细胞株及其构建方法和应用 | |
CN109652451A (zh) | 一种绵羊羊睾丸细胞永生化细胞系的构建方法及其应用 | |
CN116064660B (zh) | 绵羊诱导性多能干细胞及其制备方法 | |
CN114196703B (zh) | 一种提高牦牛克隆胚胎发育率的载体、细胞和方法 | |
CN111454944B (zh) | 一种分离的rna及其dna模板的合成方法 | |
CN112175995B (zh) | 一种vsx2绿色荧光报告基因载体系统及其构建方法 | |
CN115896109A (zh) | 一种基于碱基编辑器ABE系统构建ABO基因点突变的hiPS细胞系的构建方法 | |
CN105063057A (zh) | 锌指核酸酶介导的猪mstn基因突变序列及其应用 | |
CN116948979A (zh) | pAPN突变体和用于pAPN基因定点修饰的系统及应用 | |
CN115725578A (zh) | 一种高效编辑smn2基因的单碱基编辑系统及其应用 | |
CN116042716A (zh) | 一种Cre重组酶调控系统及其应用 | |
CN114457080A (zh) | 一种ARHGAP11B基因缺失的iPSC细胞系的构建方法及应用 |
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 |