CN114561408B - Construction method and application of retinal pigment degeneration disease model - Google Patents
Construction method and application of retinal pigment degeneration disease model Download PDFInfo
- Publication number
- CN114561408B CN114561408B CN202210078073.7A CN202210078073A CN114561408B CN 114561408 B CN114561408 B CN 114561408B CN 202210078073 A CN202210078073 A CN 202210078073A CN 114561408 B CN114561408 B CN 114561408B
- Authority
- CN
- China
- Prior art keywords
- wtap
- gene
- exon
- retinal
- retinal pigment
- 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
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 55
- 201000007737 Retinal degeneration Diseases 0.000 title claims abstract description 41
- 238000010276 construction Methods 0.000 title abstract description 15
- 210000004027 cell Anatomy 0.000 claims abstract description 32
- 210000001525 retina Anatomy 0.000 claims abstract description 32
- 210000000880 retinal rod photoreceptor cell Anatomy 0.000 claims abstract description 12
- 241000699670 Mus sp. Species 0.000 claims description 26
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 17
- 238000005516 engineering process Methods 0.000 claims description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 108091033409 CRISPR Proteins 0.000 claims description 10
- 108700024394 Exon Proteins 0.000 claims description 9
- 238000003209 gene knockout Methods 0.000 claims description 9
- 238000011813 knockout mouse model Methods 0.000 claims description 9
- 230000013011 mating Effects 0.000 claims description 9
- 238000010354 CRISPR gene editing Methods 0.000 claims description 4
- 108020005004 Guide RNA Proteins 0.000 claims description 4
- 238000010362 genome editing Methods 0.000 claims description 4
- 238000010459 TALEN Methods 0.000 claims description 3
- 210000001161 mammalian embryo Anatomy 0.000 claims description 3
- 108020004999 messenger RNA Proteins 0.000 claims description 3
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 claims description 2
- 235000013601 eggs Nutrition 0.000 claims description 2
- 210000004291 uterus Anatomy 0.000 claims description 2
- 101150104379 WTAP gene Proteins 0.000 abstract description 74
- 241001465754 Metazoa Species 0.000 abstract description 29
- 230000002207 retinal effect Effects 0.000 abstract description 12
- 241000124008 Mammalia Species 0.000 abstract description 9
- 230000007850 degeneration Effects 0.000 abstract description 9
- 230000030833 cell death Effects 0.000 abstract description 4
- 230000006378 damage Effects 0.000 abstract description 3
- 230000008034 disappearance Effects 0.000 abstract description 3
- 231100000915 pathological change Toxicity 0.000 abstract description 3
- 230000036285 pathological change Effects 0.000 abstract description 3
- 108091008695 photoreceptors Proteins 0.000 abstract description 3
- 230000000750 progressive effect Effects 0.000 abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 18
- 210000005252 bulbus oculi Anatomy 0.000 description 16
- 229940079593 drug Drugs 0.000 description 16
- 239000003814 drug Substances 0.000 description 16
- 201000010099 disease Diseases 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 101000914035 Homo sapiens Pre-mRNA-splicing regulator WTAP Proteins 0.000 description 9
- 102100026431 Pre-mRNA-splicing regulator WTAP Human genes 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 208000007014 Retinitis pigmentosa Diseases 0.000 description 7
- 210000004087 cornea Anatomy 0.000 description 7
- 230000004300 dark adaptation Effects 0.000 description 6
- 210000001508 eye Anatomy 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 5
- 230000004438 eyesight Effects 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 239000008096 xylene Substances 0.000 description 5
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical group [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 4
- 239000012188 paraffin wax Substances 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 206010002091 Anaesthesia Diseases 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000037005 anaesthesia Effects 0.000 description 3
- 238000009395 breeding Methods 0.000 description 3
- 230000001488 breeding effect Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 238000011532 immunohistochemical staining Methods 0.000 description 3
- 230000004301 light adaptation Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 210000000608 photoreceptor cell Anatomy 0.000 description 3
- 239000000049 pigment Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- NCYCYZXNIZJOKI-IOUUIBBYSA-N 11-cis-retinal Chemical compound O=C/C=C(\C)/C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-IOUUIBBYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 102000016397 Methyltransferase Human genes 0.000 description 2
- 108060004795 Methyltransferase Proteins 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 102000004330 Rhodopsin Human genes 0.000 description 2
- 108090000820 Rhodopsin Proteins 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 229940000406 drug candidate Drugs 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 238000012744 immunostaining Methods 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 230000011987 methylation Effects 0.000 description 2
- 238000007069 methylation reaction Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 230000003950 pathogenic mechanism Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 238000011830 transgenic mouse model Methods 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 201000004569 Blindness Diseases 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 1
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 1
- 206010018341 Gliosis Diseases 0.000 description 1
- 241000282575 Gorilla Species 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 102000005882 Histone H2a Dioxygenase AlkB Homolog 1 Human genes 0.000 description 1
- 108010005336 Histone H2a Dioxygenase AlkB Homolog 1 Proteins 0.000 description 1
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 1
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 1
- 101100066746 Mus musculus Wtap gene Proteins 0.000 description 1
- 208000006550 Mydriasis Diseases 0.000 description 1
- 208000001140 Night Blindness Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 206010057430 Retinal injury Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 239000008049 TAE buffer Substances 0.000 description 1
- 206010047571 Visual impairment Diseases 0.000 description 1
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- HGEVZDLYZYVYHD-UHFFFAOYSA-N acetic acid;2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid Chemical compound CC(O)=O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O HGEVZDLYZYVYHD-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 210000005069 ears Anatomy 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000002001 electrophysiology Methods 0.000 description 1
- 230000007831 electrophysiology Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- DZGCGKFAPXFTNM-UHFFFAOYSA-N ethanol;hydron;chloride Chemical compound Cl.CCO DZGCGKFAPXFTNM-UHFFFAOYSA-N 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 1
- 230000007387 gliosis Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 238000003801 milling Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000002637 mydriatic agent Substances 0.000 description 1
- 201000008026 nephroblastoma Diseases 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 230000025308 nuclear transport Effects 0.000 description 1
- 230000008212 organismal development Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000004254 retinal expression Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 208000029257 vision disease Diseases 0.000 description 1
- 230000004393 visual impairment Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 125000002256 xylenyl group Chemical class C1(C(C=CC=C1)C)(C)* 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
- C12N9/1007—Methyltransferases (general) (2.1.1.)
-
- 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
- A01K67/0276—Knock-out vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- 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
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- 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
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- 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
-
- 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
-
- 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)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Veterinary Medicine (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Environmental Sciences (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Molecular Biology (AREA)
- Rheumatology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Public Health (AREA)
- Plant Pathology (AREA)
- Epidemiology (AREA)
- Urology & Nephrology (AREA)
- Toxicology (AREA)
- Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Endocrinology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Diabetes (AREA)
- Medicinal Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a construction method and application of a retinal pigment degeneration disease model, and relates to the technical field of medical engineering. It comprises the following steps: knocking out Wtap gene sequence in human target mammal retina rod cell genome. The target animal from which the Wtap gene is knocked out can be caused to exhibit characteristics associated with retinal pigment degeneration disease by knocking out the Wtap gene sequence in the genome of the non-human target mammalian retinal rod cell. Such as rod cell death, is mainly manifested by photoreceptor damage, degeneration, progressive thinning of the outer retinal nuclear layer until disappearance, and corresponding pathological changes in the outer retinal network and other relevant cell layers. Thus, a non-human target mammal in which the Wtap gene in the retinal rod cells is knocked out can be used as a model of retinal pigment degeneration disease.
Description
Technical Field
The invention relates to the technical field of medical engineering, in particular to a construction method and application of a retinal pigment degeneration disease model.
Background
Retinal pigment degeneration (Retinitis pigmentosa, RP) is a hereditary blinding fundus disease in which degeneration of photoreceptor cells and pigment epithelium occurs together, and is characterized by degeneration of rod cells, further, the cone cells are affected, and finally, all photoreceptor cells are degenerated. Wherein a typical RP patient exhibits a generalized impairment of rod cells; atypical RP patients are predominantly cone-cell impaired. Typical RP patients initially develop night blindness due to damaged rod cells, gradually diminishing vision, and eventually blindness.
However, there are still many difficulties in diagnosing and treating RP, mainly due to the high heterogeneity of clinical phenotypes and genetics, and the lack of study of pathogenic mechanisms, which results in unclear specific molecular mechanisms of RP, which presents a great impediment to the diagnosis and treatment of RP, and the need to conduct intensive study on detailed pathogenic mechanisms of RP, so that constructing a corresponding RP disease model becomes particularly important.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a construction method and application of a retinal pigment degeneration disease model to solve the technical problems.
Wtap (Wilms tumours 1-associating protein, MGI: 1926395), this gene is located on mouse chromosome 17 at 12966799-12992543bp, full length 25.7kb, and its cDNA at full length 4967bp, contains 8 exons. Previous studies have shown that Wtap is involved in the m6A methylation modification process of RNA as part of the methyltransferase complex, which is widely distributed in various types of tissue cells. The formation process of m6A methylation mainly comprises methyltransferase complex (METTL 3, METT14, WTAP and the like), demethylase (FTO and ALKBH 5) and reading protein (YTDDF 1/2/3, YTDYC 1/2) for dynamic regulation and control, and is closely related to gene expression regulation and control. Second, m6A may be involved in biological processes such as mRNA transcription, selective cleavage, nuclear transport, translation, and degradation, resulting in RNA dysfunction, which in turn affects a range of animal vital activities. At present, the research on WTAP protein functions is gradually increased, mainly comprising the influences on tumorigenesis, organism development and the like, but the detailed action mechanism and the biological functions thereof in retina are not obvious, so that the development and application of the WTAP protein are limited. Therefore, the WTAP has great potential for treating and discussing the etiology of the retinal pigment degeneration disease.
The invention is realized in the following way:
the invention provides a construction method of a retinal pigment degeneration disease model, which comprises the following steps: knocking out Wtap gene sequence in human target mammal retina rod cell genome.
The inventors found that the target animals with the Wtap gene knocked out can be caused to exhibit characteristics associated with retinal pigment degeneration disease by knocking out the Wtap gene sequence in the genome of the non-human target mammalian retinal rod cells. Such as rod cell death, is mainly manifested by photoreceptor damage, degeneration, progressive thinning of the outer retinal nuclear layer until disappearance, and corresponding pathological changes in the outer retinal network and other relevant cell layers. Thus, animals in which the Wtap gene in the retinal rod cells is knocked out can be used as a model of retinal pigment degeneration disease.
The model of the retinal pigment degeneration disease provided by the invention can be used in the fields of retinal pigment degeneration disease research and the like, provides a new model for researching the disease such as pathogenesis, mechanism and screening related medicines, and provides a good model foundation for deeply researching the treatment and etiology discussion of WTAP on the retinal pigment degeneration disease.
In a preferred embodiment of the present invention, the above-mentioned Wtap gene sequence in the genome of the target human mammalian retina rod cell is: the exon sequence of Wtap gene is knocked out.
The above-described Wtap gene sequences in the genome of the human target mammalian retinal rod cell include, but are not limited to, the full-length sequence of the knockdown Wtap gene, and partial sequences (e.g., partial exon sequences) of the knockdown Wtap gene. Regardless of the type of sequence knocked out (partial or full length), it is within the scope of the present invention that the expression of the Wtap gene is silenced in rod cells, so that the animal exhibits the corresponding characteristics of a retinitis pigmentosa disease.
In a preferred embodiment of the present invention, the above-mentioned Wtap gene sequence in the genome of the target human mammalian retina rod cell is: knocking out the sequence of at least one of the 1 st exon to the 8 th exon of the Wtap gene.
Such as the 1 st, 2 nd, 3 rd, 4 th, 5 th, 6 th, 7 th or 8 th exons, e.g.the 1 st, 2 nd and 3 rd exons are knocked out simultaneously.
In a preferred embodiment of the present invention, the above-mentioned Wtap gene sequence in the genome of the target human mammalian retina rod cell is: the sequence in exon 3 of the Wtap gene or the exon combination consisting of the exon 3 and other exons is knocked out.
The other exons are selected from at least one of the following exons: exon 1, exon 2, exon 4, exon 5, exon 6, exon 7 and exon 8.
In a preferred embodiment of the present invention, the gene editing technique used for the knockout includes: at least one of CRISPR/Cas9 technology, artificial nuclease-mediated zinc finger nuclease technology (ZFN), transcription activator-like effector nuclease technology (transceription activator-like effector nucleases, TALEN), and Cre-loxp gene knockout technology.
In a preferred embodiment of the present invention, the gene editing technique used for the knockout includes: CRISPR/Cas9 technology and Cre-loxp gene knockout technology.
In a preferred embodiment of the application of the present invention, the above construction method includes: transforming a donor vector containing gRNA of a Wtap gene of a non-human target mammal and Cas9 mRNA into fertilized eggs of the non-human target mammal, taking embryo cells to be transplanted into uterus of a pseudo-pregnant non-human target mammal to obtain a first-established animal with the Wtap gene conditionally knocked out, mating the first-established animal to obtain a retina rod cell Wtap conditionally knocked-out animal;
the gRNA has a loxP site.
The above-mentioned mating of the first-established animal to obtain the retina rod cell Wtap conditional knockdown animal includes:
mating and breeding the first-established animal and a target wild animal to obtain a heterozygote Wtap gene conditional knockout animal; and then mating and breeding the obtained heterozygote Wtap gene conditional knockout animals to obtain the Wtap gene conditional knockout homozygote animals.
Mating the obtained Wtap gene conditional knockout homozygote animal with the Rod-Cre gene transferred animal to obtain a retina Rod cell Wtap knockout gene animal, and the retina Rod cell Wtap knockout homozygote animal can be used as a retina pigment degeneration disease model.
Rod-Cre transgenic animals (mice) Rod-Cre transgenic mice (stock number: 015850) were purchased from Jackson laboratories (Jackson Laboratory). Line name full: B6.Cg-Pde6b+Tg (Rho-ice) 1Ck/Boc.
In a preferred embodiment of the present invention, the non-human target mammal is selected from any one of a mouse, a rat, a dog, a pig, a monkey, and a ape. Any animal can be selected as long as it is an animal having the Wtap gene, and it is intended to be a target (non-human mammal) in the above-described construction method of the present invention, and it is within the scope of the present invention to knock out the Wtap gene in the rod cells thereof, thereby exhibiting disease characteristics of retinitis pigmentosa, and to use it as a model of retinitis pigmentosa disease in the field of retinitis pigmentosa disease research.
In other embodiments, the non-human target mammal is selected from sheep or gorilla.
The invention also provides application of the retinal pigment degeneration disease model constructed by the construction method of the retinal pigment degeneration disease model in screening medicines for preventing or treating retinal pigment degeneration diseases.
The vision of the model of the retinitis pigmentosa disease is improved after administration of the candidate drug compared to prior to administration of the candidate drug; the candidate drug is indicated as a drug for preventing or treating a retinal pigment degeneration disease.
If the drug candidate is administered, the extraretinal nucleus layer thickness is thicker or has a tendency to thicken compared to before administration of the drug candidate; the candidate drug is indicated as a drug for preventing or treating a retinal pigment degeneration disease.
If the candidate drug is administered, the outer retinal segment increases compared to prior to administration of the candidate drug; the candidate drug is indicated as a drug for preventing or treating a retinal pigment degeneration disease.
The invention also provides application of the retinal pigment degeneration disease model constructed by the construction method of the retinal pigment degeneration disease model in retinal pigment degeneration disease research, wherein the research aims at diagnosis or treatment of non-diseases.
The disease model obtained by the construction method has typical characteristics of the retinal pigment degeneration disease, has very wide application prospect, and for example, the disease model is used for researching the pathogenesis of the retinal pigment degeneration disease, thereby providing a foundation for deep understanding of the research of the retinal pigment degeneration disease. Or it can be used for screening drugs for preventing or treating retinal pigment-modified diseases, evaluating the efficacy or prognosis of drugs, etc.
The invention has the following beneficial effects:
the inventors found that the target animals with the Wtap gene knocked out can be caused to exhibit characteristics associated with retinal pigment degeneration disease by knocking out the Wtap gene sequence in the genome of the non-human target mammalian retinal rod cells. Such as rod cell death, is mainly manifested by photoreceptor damage, degeneration, progressive thinning of the outer retinal nuclear layer until disappearance, and corresponding pathological changes in the outer retinal network and other relevant cell layers. Thus, a non-human target mammal in which the Wtap gene in the retinal rod cells is knocked out can be used as a model of retinal pigment degeneration disease.
The construction of the model of the retinal pigment degeneration disease provides a good model foundation for further researching the treatment and etiology discussion of the WTAP on the retinal pigment degeneration disease, and also provides a good application prospect for screening medicines for preventing or treating the retinal pigment degeneration disease.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows that the retina rod cell specifically knocks out Wtap gene mice (Wtap RKO ) A construction flow chart and a genotype identification result chart;
FIG. 2 is a graph showing the detection result of Wtap gene knockout efficiency;
FIG. 3 is a diagram of Electroretinogram (ERG) test results;
FIG. 4 is a graph showing immunohistochemical staining results of retinal sections of mice with specific Wtap gene knocked out by retinal rod cells;
FIG. 5 is a graph showing IHC staining results of mice specifically knocked out Wtap gene by 6 month old retinal rod cells;
FIG. 6 is a graph showing IHC staining results of mice with Wtap gene specifically knocked out by 8 month old retinal rod cells.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
In this embodiment, a mouse is used as a target animal, and the construction method of the retinal pigment degeneration disease model provided by the invention is described, wherein the Wtap gene knockout route is shown in fig. 1 a, and the specific operation is as follows:
1) Wtap gene conditional knockdown model (https:// www.modelorg.com; line name: c57BL/6-Wtapem1 (flox) Smoc, catalog number: NM-CKO-190009) was purchased from Shanghai, south mode Biotech Co., ltd. The loxp sequences arranged in the same direction are placed at two ends of the No. 3 exon of the Wtap gene.
2) Mating and breeding the heterozygote mice with the Wtap gene subjected to conditional knockout obtained in the step 1) to obtain homozygous mice with the Wtap gene subjected to conditional knockout.
3) Mating the homozygote mouse with the Wtap gene conditional knocked out in the step 2) with the mouse with the Rod-Cre gene transferred to obtain the Wtap knocked out mouse of the retina Rod cell, and the Wtap knocked out mouse can be used as a retina pigment degeneration disease model. Rod-Cre transgenic mice (line name: B6.Cg-Pde6b+Tg (Rho-ice) 1 Ck/Boc) from Jackson laboratories; stock number 015850).
Carrying out genotype identification on the obtained offspring mice, wherein the identification method is as follows:
1) Cutting a small amount of tissue samples from the tail tips of the mice, and placing the small tissue samples into a clean 1.5ml centrifuge tube;
2) 100 μl of lysate (40mM NaOH,0.2mM EDTA solution) was added to the centrifuge tube and heated at 100deg.C in the metal bath for 1h;
3) The tube was removed, cooled to room temperature, 100. Mu.l of a neutralization solution (40 mM Tris-HCl, pH 5.5) was added, and 10000g of the tube was centrifuged for 2 minutes, and the supernatant was used for genotyping of mice.
4) And (3) PCR amplification: the reaction system is that
2×Taq Mix 10μL
Tissue lysate 4. Mu.L
Primer 1 (Wtap-loxP-Forward or Rod-Cre-F), 1. Mu.L (concentration: 10 mM)
Primer 2 (Wtap-loxP-Reverse or Rod-Cre-R), 1. Mu.L (concentration: 10 mM)
ddH 2 O 4μL。
The primer sequences were as follows:
Wtap-loxP-F：5’-TTCATAACGATTTCTCTGTG-3’；
Wtap-loxP-R：5’-AGTTCGCAATACCTAATCA-3’；
Rod-Cre-F：TCAGTGCCTGGAGTTGCGCTGTGG；
Rod-Cre-R：CTTAAAGGCCAGGGCCTGCTTGGC。
amplification procedure:
after the PCR reaction system is prepared, the template DNA is fully denatured by preheating for 5 minutes at 95 ℃ on a PCR instrument, and then the amplification cycle is carried out. In each cycle, the template is denatured prior to maintaining at 95 ℃ for 30 seconds, then the temperature is reduced to the renaturation temperature of 60 ℃ for 30 seconds, and the primer and the template are fully annealed; the reaction was kept at 72℃for 45 seconds, and primers were allowed to extend on the template to synthesize DNA, thereby completing one cycle. This cycle was repeated 25 times to accumulate a large amount of amplified DNA fragments. Finally, the product was left to extend intact at 72℃for 5 minutes and stored at 4 ℃.
5) Gel electrophoresis.
1g of agarose was weighed and placed in 100ml of TAE buffer, and melted in a microwave oven to prepare 1% agarose gel. 10ul of the PCR product was taken in the wells and subjected to 120V constant pressure agarose electrophoresis for 15min. Imaging was performed with a gel imaging system.
Shown in fig. 1B: genotyping of Wtap-specific knockout miceAs a result, wtap +/+ Control mice (labeled Wtap Ctrl ) The stripe size is 351p; wtap flox/+ The method comprises the steps of carrying out a first treatment on the surface of the Rod-Cre represents heterozygote obtained after mating a Wtap gene conditional knockout heterozygote mouse with a Rod-Cre gene transferred mouse, and the heterozygote has two bands 351bp and 385bp; wtap flox/flox The method comprises the steps of carrying out a first treatment on the surface of the Rod-Cre represents a Wtap knockout homozygous mouse (labeled Wtap RKO ) The band size was 385bp, which has a loxp site. Based on the results shown in FIG. 1B, the identification method used in this example can effectively identify the genotype of the newborn mice for subsequent study.
Example 2
In the embodiment, the Rod-Cre knockout mouse Wtap is analyzed by adopting an immunoblotting (Western blot) experiment RKO Gene knockout efficiency in retina.
The method comprises the following steps:
1) Control and knockout mouse retinas were obtained, respectively, and 200ul of protein lysate RIPA was added after sufficient milling.
2) After sonicating the cells, they were lysed on ice for 20min.
3) After centrifugation at 16000g for 10min at 4℃the supernatant was transferred to another clean centrifuge tube, 50. Mu.l of protein loading solution was added and mixed well and heated at 95℃for 5min.
4) After the sample was cooled, 20. Mu.l of each sample was subjected to polyacrylamide gel electrophoresis (SDS-PAGE) at 160V to separate proteins.
5) After SDS-PAGE is finished, cutting a nitrocellulose membrane with proper size according to the requirement, paving filter paper, glue, the nitrocellulose membrane and the filter paper in sequence, removing bubbles, putting the membrane transferring groove into an ice-water bath, and transferring the membrane by adopting a constant current of 0.28A for 2 hours.
6) After the transfer of the film is finished, the nitrocellulose film is washed once by pure water, dried and marked. Then blocked with 8% skim milk for 2h.
7) After blocking was completed, a certain amount of primary antibody diluted in blocking solution in a certain proportion (according to the instructions for antibody use) was added and incubated overnight at 4 ℃.
8) The primary antibody was recovered, washed with 1 XTBE buffer 4 times for 10min each, and appropriate secondary antibody was selected depending on the source of primary antibody, and horseradish peroxidase (HRP) -labeled secondary antibody was diluted with 1 XTBE and incubated on a shaker at room temperature for 2h.
9) After the secondary antibody incubation was completed, the membrane was washed 3 times with 1 XTBST for 10min each, and the protein was detected with Thermo ELC luminescence kit using a Bio-Rad chemiluminescent gel imaging system.
The results are shown in FIG. 2. At 6 months of age in mice WTAP disappeared in retinal expression, and statistics indicate that WTAP expression was significantly reduced in knockout mouse retinal rod cells, indicating that it was knocked out.
Example 3
Wtap knockout mice (Wtap) RKO ) Performing ERG vision test:
1) Dark adaptation animals should adapt overnight, and the environment should be absolutely clear;
2) The next day of anesthesia: weighing and injecting into abdominal cavity; deep anesthesia is preferred;
3) Animal fixation and mydriasis: after anesthesia was completed, mice were fixed with tape in front of animal test platform under dark red light: the mice are required to be ensured to lie on the ground, namely, the eyes are consistent in height relative to the stimulation port of the flash stimulator, and are fully exposed, and mydriatic agent is dripped.
4) Electrode installation: preheating a retinogram instrument (Espion Visual Electrophysiology System, diagnosyllc, lt-tleton, MA, USA), coating conductive paste on the electrode, clamping the tail of a mouse, and inserting the mouse into an amplifier ground interface; the double-ended needle electrode is inserted into the back cervical skin (approximately in the middle of two ears) and is simultaneously connected with the negative interfaces of the two channels; the gold ring electrode was clamped on the electrode holder of the animal experiment platform, the angle was carefully adjusted, and the center tip of the cornea was slightly contacted. One channel positive electrode is connected with the right eye, and the two channel positive electrodes are connected with the left eye. The contact effect of the gold ring electrode and cornea is improved by dropping normal saline to eyes through the needle tube. The two gold ring electrodes are ensured to contact the same position of the center positive end of the cornea of two eyes at the same angle and in the same mode.
5) And closing the dark red light after the record oscillographic signal confirms no error. It can be tried to record the dark adaptation light intensity of 3.0 cd/s.m 2 ERG detection of (C)Confirm the quality of the following signal: if the amplitude of the eyes is greatly different from that expected, it is recommended to check the mounting position of the gold ring electrode again. Then sequentially recording dark adaptation light intensity of 3.0/10.0 cd/s.m 2 After recording the system will automatically turn on the backlight.
6) Continuously recording the light adaptation light intensity of 10.0 cd/s.m 2 Is a signal of (a).
The results found that at 6 months, both the a-wave and b-wave of the knockout mice were significantly reduced under dark adaptation conditions compared to the control mice, indicating that Wtap resulted in impaired vision after rod cell knockout (fig. 3).
In fig. 3: a, B, a retina electrogram waveform of a Wtap control mouse under dark adaptation under different light intensities;
c, D at 3.0 cd/s.m 2 The Wtap knockout mice are subjected to electroretinogram waveforms under light adaptation under light intensity;
e, F: dark adaptation of 3.0 and 10.0 cd/s.m 2 Light intensity and light adaptation of 10.0cd/s/m 2 The statistics of a wave and b wave of light intensity shows that the dim eyesight of the knockout mouse is obviously reduced.
Example 4
The present example performs H & E staining of retinal paraffin sections.
Retinas of 6,9 month old mice were stained by paraffin section, hematoxylin-eosin staining (H & E staining method) as follows:
1) Quickly taking eyeball tissues of a mouse, and placing the eyeball tissues in a fixing solution for fixing for 24 hours;
2) Embedding paraffin, slicing with thickness of 4 μm;
3) Slices were conventionally dewaxed with xylene, washed with multi-stage ethanol to water: xylene (I) 5 min- & gt xylene (II) 5 min- & gt 100% ethanol 2 min- & gt 95% ethanol 1 min- & gt 80% ethanol 1 min- & gt 75% ethanol 1 min- & gt distilled water washing 2min;
4) Hematoxylin staining for 5 minutes, washing with tap water;
5) Ethanol hydrochloride differentiation for 30 seconds;
6) Soaking in tap water for 15 minutes;
7) And (5) placing eosin solution for 2 minutes.
8) Conventional dehydration, transparency and sealing sheet: 95% ethanol (I) 1min, 95% ethanol (II) 1min, 100% ethanol (I) 1min, 100% ethanol (II) 1min, xylenol carbonic acid (3:1) 1min, xylene (I) 1min, xylene (II) 1min and neutral resin sealing.
9) And photographing under a microscope.
As a result, it was found that at 6,9 months, compared with Wtap Ctrl (control) mice, wtap RKO The extraretinal nuclear layer thickness of (knocked out) mice was significantly thinner, indicating photoreceptor cell death (fig. 4).
Fig. 4 a: h & E staining results of the mouse retina paraffin sections with the Wtap genes knocked out by the retina rod cells specifically show that the outer nuclear layer and the inner nuclear layer are thin;
b: statistics of the thickness of the Outer Nuclear Layer (ONL) of the retina of Wtap knockout mice at different sites.
Example 5
Immunostaining of frozen sections of retina: after the retina rod cells constructed in the example 1 with the age of 6 months are taken to specifically knock out the Wtap gene, the mice are killed after neck breaking, eyeballs are quickly taken and put into 4% PFA, the eyeballs are fixed on ice for 15min, then the eyeballs are cut on cornea, and then the fixation on ice is continued. After 2h, PBS buffer was washed 3 times, then the eyeball was dehydrated in 30% sucrose solution for 2h, then the cornea and crystals were cut off under a dissecting scope, OCT was embedded and rapidly frozen in a refrigerator at-80 ℃. After about 10min, the OCT embedded eyeball is taken out, and the eyeball is placed in a frozen microtome for balancing at the temperature of minus 25 ℃ for about 30min, and then the eyeball can be sectioned. The slice thickness was 12. Mu.m.
After slicing, higher quality pieces were selected and placed in an oven at 37 ℃ for 30min, then an immunohistochemical pen was circled around the area with retinal tissue, washed three times with PBS to remove OCT, then 5% NDS (containing 0.25% triton) were blocked through for 2h, primary antibodies were incubated, and overnight at 4 ℃. The following day, after three times of PBS washing, the corresponding fluorescent secondary antibodies are incubated, and then three times of PBS washing are carried out, and the plates are sealed and observed.
The results are shown in FIG. 5. Marking the outer section of the rod by using a Rhodopsin antibody at the age of 6 months of mice, and counterstaining the nucleus by using DAPI; the outer segment antibody Rhodopsin was stained by frozen tissue sections of the retina, and compared with Wtap Ctrl (control)Mouse, wtap RKO The outer segments of the retinas of (knockdown) mice were significantly shortened and a significant degeneration characterization occurred.
Example 6
Immunostaining of frozen sections of retina: after the retina rod cells constructed in the example 1 with the age of 8 months are taken to specifically knock out the Wtap gene, the mice are killed after neck breaking, eyeballs are quickly taken and put into 4% PFA, the eyeballs are fixed on ice for 15min, then the eyeballs are cut on cornea, and then the fixation on ice is continued. After 2h, PBS buffer was washed 3 times, then the eyeball was dehydrated in 30% sucrose solution for 2h, then the cornea and crystals were cut off under a dissecting scope, OCT was embedded and rapidly frozen in a refrigerator at-80 ℃. After about 10min, the OCT embedded eyeball is taken out, and the eyeball is placed in a frozen microtome for balancing at the temperature of minus 25 ℃ for about 30min, and then the eyeball can be sectioned. The slice thickness was 12. Mu.m.
After slicing, higher quality pieces were selected and placed in an oven at 37 ℃ for 30min, then an immunohistochemical pen was circled around the area with retinal tissue, washed three times with PBS to remove OCT, then 5% NDS (containing 0.25% triton) were blocked through for 2h, primary antibodies were incubated, and overnight at 4 ℃. The following day, after three times of PBS washing, the corresponding fluorescent secondary antibodies are incubated, and then three times of PBS washing are carried out, and the plates are sealed and observed.
The results are shown in FIG. 6. At 8 months of age, the glial cell marker GFAP was stained by frozen tissue sections of the retina, as compared with Wtap Ctrl (control) mice, wtap RKO The (knockdown) mouse retina showed significant gliosis, and the inflammatory response was enhanced, indicating retinal damage.
In summary, it can be seen that, taking a mouse as an example, the embodiment of the invention specifically knocks out the Wtap gene in the retina rod cells thereof by using a gene knockout technology (Cre-loxP knockout technology), so that the mouse shows typical characteristics of retinal pigment degeneration diseases such as visual impairment, shortening and degeneration of extracellular nodes, loss of visual cells and the like. Thus, it was fully demonstrated that conditional knockout of Wtap gene in rod cells of retina can cause target animals to exhibit retinitis pigmentosa disease. The construction of the model of the retinal pigment degeneration disease provides a good model foundation for further researching the treatment and etiology discussion of the WTAP on the retinal pigment degeneration disease, and also provides a good application prospect for screening medicines for preventing or treating the retinal pigment degeneration disease.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A method for constructing a model of a retinal pigment degeneration disease, comprising: knockout of mouse retinal rod cell genomeWtapThe 3 rd exon of the gene, or the sequence in the exon combination consisting of the 3 rd exon and other exons is knocked out, wherein the other exons are selected from at least one of the following exons: exon 1, exon 2, exon 4, exon 5, exon 6, exon 7 and exon 8.
2. The method for constructing a model of a retinal pigment degeneration disease according to claim 1, wherein said knockout adopts a gene editing technique comprising: at least one of CRISPR/Cas9 technology, ZFN technology, TALEN technology, and Cre-loxp gene knockout technology.
3. The method for constructing a model of a retinal pigment degeneration disease according to claim 2, wherein the gene editing technique used for knockout includes: CRISPR/Cas9 technology and Cre-loxp gene knockout technology.
4. A method of constructing a model of a retinal pigment degeneration disease according to claim 3, characterized in that the method of constructing comprises: will contain miceWtapTransforming the donor vector of the gRNA of the gene and the Cas9 mRNA into fertilized eggs of mice, taking embryo cells and transplanting the embryo cells into uterus of pseudopregnant mice to obtainWtapA first-established mouse with conditional gene knockout, mating the first-established mouse to obtain retina visionStem cellsWtapConditional knockout mice;
the gRNA has a loxP site.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210078073.7A CN114561408B (en) | 2022-01-24 | 2022-01-24 | Construction method and application of retinal pigment degeneration disease model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210078073.7A CN114561408B (en) | 2022-01-24 | 2022-01-24 | Construction method and application of retinal pigment degeneration disease model |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114561408A CN114561408A (en) | 2022-05-31 |
| CN114561408B true CN114561408B (en) | 2023-04-21 |
Family
ID=81714798
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210078073.7A Active CN114561408B (en) | 2022-01-24 | 2022-01-24 | Construction method and application of retinal pigment degeneration disease model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114561408B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115176760B (en) * | 2022-07-07 | 2023-11-17 | 电子科技大学 | Method for constructing retinal pigment degeneration disease model, application and breeding method |
| CN114916502B (en) * | 2022-07-07 | 2023-06-16 | 电子科技大学 | Construction method and application of retinal pigment degeneration disease model |
| CN116738352B (en) * | 2023-08-14 | 2023-12-22 | 武汉大学人民医院（湖北省人民医院） | Method and device for classifying abnormal rod cells of retinal vascular occlusion disease |
| CN117230077B (en) * | 2023-11-13 | 2024-02-23 | 四川省医学科学院·四川省人民医院 | Application of Hakai gene in RP disease model construction and construction method |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108029638B (en) * | 2017-12-04 | 2020-04-21 | 四川省人民医院 | Construction method and application of animal model of retinitis pigmentosa disease |
| US11653636B2 (en) * | 2019-11-15 | 2023-05-23 | University Of Ulsan Foundation For Industry Cooperation | Method of making a rat model of retinal degeneration and rat model made thereby |
| CN112715484B (en) * | 2020-12-29 | 2022-04-22 | 四川省人民医院 | Method for constructing retinal pigment degeneration disease model, application and breeding method |
| CN113621649B (en) * | 2021-09-14 | 2023-05-26 | 商丘市第一人民医院 | Construction method and application of retinal pigment degeneration disease model |
-
2022
- 2022-01-24 CN CN202210078073.7A patent/CN114561408B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN114561408A (en) | 2022-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114561408B (en) | Construction method and application of retinal pigment degeneration disease model | |
| CN113621649B (en) | Construction method and application of retinal pigment degeneration disease model | |
| CN114916502B (en) | Construction method and application of retinal pigment degeneration disease model | |
| CN112715484B (en) | Method for constructing retinal pigment degeneration disease model, application and breeding method | |
| CN111269945B (en) | Method for constructing retinal pigment degeneration disease model by using Gm20541 gene and application | |
| Rachel et al. | Combining Cep290 and Mkks ciliopathy alleles in mice rescues sensory defects and restores ciliogenesis | |
| CN115176760B (en) | Method for constructing retinal pigment degeneration disease model, application and breeding method | |
| US20200375157A1 (en) | Construction method for animal model for retinitis pigmentosa diseases and application | |
| CN109182355B (en) | The construction method of retinal neovascularization disease model and application | |
| CN111500640B (en) | Construction method and cultivation method of RP disease model | |
| Narfström et al. | The domestic cat as a large animal model for characterization of disease and therapeutic intervention in hereditary retinal blindness | |
| CN107586791B (en) | A kind of construction method of incoordination animal model and application | |
| CN111484981B (en) | Method for constructing retinal neovascular disease model and application | |
| CN112980879B (en) | Construction method and application of retinal vascular disease model | |
| Dai et al. | Photoreceptor degeneration in a new Cacna1f mutant mouse model | |
| CN111778281B (en) | Construction method and application of retina bipolar cytopathy model | |
| CN117296799B (en) | Construction method and application of retinal pigment degeneration disease model | |
| CN108315350B (en) | COX5A overexpression/BDNF low expression transgenic mouse model and construction method and application thereof | |
| CN108396036B (en) | Over-expression COX5A transgenic mouse model and construction method and application thereof | |
| CN117230077B (en) | Application of Hakai gene in RP disease model construction and construction method | |
| CN117305364A (en) | Method for constructing retina degeneration disease model by using gene manipulation technology and application | |
| CN114255823A (en) | Construction method and application of retinal degenerative disease model | |
| CN116515908B (en) | Method for establishing RPE65 gene editing model dog | |
| WO2021169333A1 (en) | Use of znf124 gene in early screening or auxiliary diagnosis of retinitis pigmentosa diseases | |
| CN116103292B (en) | Construction method and application of ZDHC 21 gene mutation animal model |
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 |