CN113621649B - 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
- CN113621649B CN113621649B CN202111074309.1A CN202111074309A CN113621649B CN 113621649 B CN113621649 B CN 113621649B CN 202111074309 A CN202111074309 A CN 202111074309A CN 113621649 B CN113621649 B CN 113621649B
- Authority
- CN
- China
- Prior art keywords
- disease
- model
- retinal pigment
- ythdf1
- gene
- 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 68
- 201000007737 Retinal degeneration Diseases 0.000 title claims abstract description 30
- 238000010276 construction Methods 0.000 title claims abstract description 17
- 239000003814 drug Substances 0.000 claims abstract description 26
- 229940079593 drug Drugs 0.000 claims abstract description 25
- 230000002207 retinal effect Effects 0.000 claims abstract description 24
- 201000010099 disease Diseases 0.000 claims abstract description 23
- 208000007014 Retinitis pigmentosa Diseases 0.000 claims abstract description 17
- 238000012216 screening Methods 0.000 claims abstract description 9
- 241000699670 Mus sp. Species 0.000 claims description 35
- 238000005516 engineering process Methods 0.000 claims description 17
- 238000003209 gene knockout Methods 0.000 claims description 17
- 108090000623 proteins and genes Proteins 0.000 claims description 15
- 230000004048 modification Effects 0.000 claims description 14
- 238000012986 modification Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 12
- 238000012217 deletion Methods 0.000 claims description 11
- 230000037430 deletion Effects 0.000 claims description 11
- 230000004438 eyesight Effects 0.000 claims description 7
- 108091033409 CRISPR Proteins 0.000 claims description 6
- 238000010362 genome editing Methods 0.000 claims description 5
- 230000006872 improvement Effects 0.000 claims description 4
- 238000010354 CRISPR gene editing Methods 0.000 claims description 2
- 238000010459 TALEN Methods 0.000 claims description 2
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 claims description 2
- 229940000406 drug candidate Drugs 0.000 claims 2
- 101100214171 Mus musculus Ythdf1 gene Proteins 0.000 abstract description 41
- 101150103569 Ythdf1 gene Proteins 0.000 abstract description 40
- 241001465754 Metazoa Species 0.000 abstract description 30
- 238000011160 research Methods 0.000 abstract description 11
- 230000030833 cell death Effects 0.000 abstract description 6
- 230000008506 pathogenesis Effects 0.000 abstract description 6
- 230000007850 degeneration Effects 0.000 abstract description 5
- 230000007246 mechanism Effects 0.000 abstract description 5
- 230000000750 progressive effect Effects 0.000 abstract description 5
- 230000006378 damage Effects 0.000 abstract description 4
- 238000011161 development Methods 0.000 abstract description 4
- 230000008034 disappearance Effects 0.000 abstract description 4
- 231100000915 pathological change Toxicity 0.000 abstract description 4
- 230000036285 pathological change Effects 0.000 abstract description 4
- 230000014509 gene expression Effects 0.000 abstract description 3
- 108091008695 photoreceptors Proteins 0.000 abstract description 3
- 230000002265 prevention Effects 0.000 abstract description 3
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 18
- 241000699666 Mus <mouse, genus> Species 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 16
- 210000001525 retina Anatomy 0.000 description 13
- 210000005252 bulbus oculi Anatomy 0.000 description 12
- 238000011813 knockout mouse model Methods 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 230000035772 mutation Effects 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 101100214180 Drosophila melanogaster Ythdf gene Proteins 0.000 description 7
- 210000004087 cornea Anatomy 0.000 description 7
- 230000004300 dark adaptation Effects 0.000 description 7
- 108700024394 Exon Proteins 0.000 description 6
- 210000001508 eye Anatomy 0.000 description 6
- 238000003780 insertion Methods 0.000 description 6
- 230000037431 insertion Effects 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 101000744742 Homo sapiens YTH domain-containing family protein 1 Proteins 0.000 description 5
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 5
- 102100039647 YTH domain-containing family protein 1 Human genes 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 239000008096 xylene Substances 0.000 description 5
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical group [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 4
- 230000004301 light adaptation Effects 0.000 description 4
- 230000013011 mating Effects 0.000 description 4
- 230000011987 methylation Effects 0.000 description 4
- 238000007069 methylation reaction Methods 0.000 description 4
- 239000012188 paraffin wax Substances 0.000 description 4
- 210000000880 retinal rod photoreceptor cell Anatomy 0.000 description 4
- 230000000007 visual effect Effects 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 3
- 108091006146 Channels Proteins 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
- 230000003321 amplification Effects 0.000 description 3
- 230000037005 anaesthesia Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 238000011532 immunohistochemical staining Methods 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 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
- 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
- 229920000936 Agarose Polymers 0.000 description 2
- 108020005004 Guide RNA Proteins 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
- 101000609949 Homo sapiens Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta Proteins 0.000 description 2
- 101000883219 Homo sapiens cGMP-gated cation channel alpha-1 Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 102000004330 Rhodopsin Human genes 0.000 description 2
- 108090000820 Rhodopsin Proteins 0.000 description 2
- 102100039174 Rod cGMP-specific 3',5'-cyclic phosphodiesterase subunit beta Human genes 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
- 238000010171 animal model Methods 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 102100038623 cGMP-gated cation channel alpha-1 Human genes 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 230000000694 effects Effects 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
- 239000006166 lysate Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 210000000608 photoreceptor cell Anatomy 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
- 238000012360 testing method Methods 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 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
- 201000004569 Blindness Diseases 0.000 description 1
- 241000283690 Bos taurus Species 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
- 241000283073 Equus caballus Species 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
- 101710088172 HTH-type transcriptional regulator RipA Proteins 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
- 101001013582 Homo sapiens N6-adenosine-methyltransferase non-catalytic subunit Proteins 0.000 description 1
- 101000914035 Homo sapiens Pre-mRNA-splicing regulator WTAP Proteins 0.000 description 1
- 101000976373 Homo sapiens YTH domain-containing protein 1 Proteins 0.000 description 1
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 102000016397 Methyltransferase Human genes 0.000 description 1
- 208000006550 Mydriasis Diseases 0.000 description 1
- 102100031578 N6-adenosine-methyltransferase non-catalytic subunit Human genes 0.000 description 1
- 208000001140 Night Blindness Diseases 0.000 description 1
- 102100027051 Nucleic acid dioxygenase ALKBH1 Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102100026431 Pre-mRNA-splicing regulator WTAP Human genes 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 206010057430 Retinal injury Diseases 0.000 description 1
- 241000168254 Siro Species 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
- 241001655798 Taku Species 0.000 description 1
- 206010045178 Tunnel vision Diseases 0.000 description 1
- 206010047571 Visual impairment Diseases 0.000 description 1
- 102100023905 YTH domain-containing protein 1 Human genes 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
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000031018 biological processes and functions 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
- 230000005779 cell damage Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 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
- 230000007547 defect Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000017858 demethylation Effects 0.000 description 1
- 238000010520 demethylation reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000004069 differentiation 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
- 235000013601 eggs Nutrition 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
- 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
- 231100000221 frame shift mutation induction Toxicity 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 239000000499 gel 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
- 238000000227 grinding Methods 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000001771 impaired effect 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
- 238000002372 labelling Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000003550 marker Substances 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
- 239000000203 mixture Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000002637 mydriatic agent Substances 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
- 230000007170 pathology 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
- 238000009877 rendering 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
- 230000002441 reversible effect Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000004296 scotopic vision Effects 0.000 description 1
- 238000012163 sequencing technique Methods 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
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 210000004291 uterus Anatomy 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
- 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
- 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
-
- 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
-
- 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
- A01K2267/0306—Animal model for genetic diseases
- A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
-
- 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
- A01K2267/0393—Animal model comprising a reporter system for screening tests
-
- 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
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Biophysics (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Behavior & Ethology (AREA)
- Physics & Mathematics (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Toxicology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a construction method and application of a retinal pigment degeneration disease model, and relates to the field of medical engineering. The construction method comprises the following steps: a model of a retinitis pigmentosa disease, which exhibits characteristics associated with the retinitis pigmentosa disease, can be obtained by preventing or inhibiting expression of the Ythdf1 gene sequence in the genome of the target animal. 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. Therefore, the novel model of the retinal pigment degeneration disease can be used in the fields of research of the retinal pigment degeneration disease and the like, and provides a richer model selection for research of the disease such as pathogenesis, mechanism and screening of related drugs. In addition, the development of the disease model provides a new target for the treatment or prevention of RP diseases.
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
Retinitis pigmentosa (Retinitis pigmentosa, RP). RP as a hereditary blinding fundus disease can be divided into two categories according to clinical phenotype: typical RP patients and atypical RP patients. Wherein, typical RP patients show common damage of the rod cells, accounting for 80% -90% of the RP patients; atypical RP patients mainly have damaged cone cells, accounting for 10% -20% of RP patients. Typical RP patients develop night blindness and progressive visual field impairment, which develop tubular vision, at the earliest, due to defects in rod cell function, until blindness. In pathology, typical RP affects mainly rod cells, causing rod cell death and secondary cone cell death, manifested by photoreceptor cell damage, degeneration, progressive thinning of the outer nuclear layer of the retina until disappearance, with corresponding pathological changes in other related cell layers of the retina.
RP has incidence rate of 1/3500 in Chinese population, and RP patients can reach hundreds of thousands of people because of the population of China, thus bringing heavy burden to families and society. At present, diagnosis and treatment of RP still face many difficulties, mainly due to their high degree of heterogeneity in clinical phenotypes and genetics, and the lack of systematic studies on their pathogenesis, leading to an unclear specific molecular mechanism of RP.
At present, a corresponding RP disease model is lacking.
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.
YTDDF 1 (YTH N6-methyladenosine RNA binding protein 1, MGI: 1917431), this gene is located on mouse chromosome 2 at 180546170-180562729bp, full length 16.56kb, and its cDNA at 3199bp, contains 5 exons.
YTDDF 1 is taken as a reading protein of m6A methylation in RNA, can identify the methylation site of the RNA and further regulate and control, and is widely distributed in various tissue cells. m6A methylation is a dynamic reversible process, the formation process mainly comprises a methyltransferase complex (METTL 3, METTL14, WTAP and the like), the demethylation process is completed by demethylases (FTO and ALKBH 5), and reading proteins (YTHDF 1-3, YTHDC1/2 and IGF2BP 1-3) are mainly responsible for identifying methylation sites and performing dynamic regulation and are closely related to gene expression regulation. Studies have shown that 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, research on YTHDF1 protein functions is mainly focused on aspects of tumorigenesis, organism development and the like, but the detailed action mechanism and the biological functions thereof in retina are not clear, so that the development and application of the YTHDF1 protein are limited. Therefore, the intensive research on the treatment and etiology of the retinal pigment degeneration disease by YTHDF1 has great potential.
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: the Ythdf1 gene sequence in the genome of the target animal is not expressed or is inhibited.
The inventors have found for the first time that a model of a retinal pigment-modified disease exhibiting characteristics associated with the retinal pigment-modified disease can be obtained such that the Ythdf1 gene sequence in the genome of the target animal is not expressed or is inhibited. 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. Therefore, the novel model of the retinal pigment degeneration disease can be used in the fields of research of the retinal pigment degeneration disease and the like, and provides a richer model selection for research of the disease such as pathogenesis, mechanism and screening of related drugs. In addition, the development of the disease model provides a new target for the treatment or prevention of RP diseases.
In a preferred embodiment of the present invention, the above construction method comprises modification by one or a combination of mutation, deletion and insertion such that the Ythdf1 gene sequence in the genome of the target animal is not expressed or is inhibited.
In one embodiment, the mutation modification means that the corresponding protein site of the Ythdf1 gene is subjected to amino acid change by the corresponding mutation to realize that the protein expressed by the Ythdf1 gene does not have a normal bioactive function or translation is terminated prematurely.
When a deletion modification is employed, it may be a deletion of one or more nucleotides, thereby rendering the Ythdf1 gene sequence non-expressed or less active. For example, deletion modification of the target gene may be achieved by deleting a part or all of the exons.
When the modification is adopted, at least one nucleotide is inserted into the Ythdf1 gene, for example, one or more nucleotides are inserted into an exon to cause frame shift mutation, so that the primary structure, the secondary structure or the tertiary structure of the expressed protein is changed, and the protein expressed by the Ythdf1 gene does not have normal biological activity function.
Therefore, whatever modification is selected, for example, combination modification, is within the scope of the present invention as long as the Ythdf1 gene sequence in the genome of the target animal is not expressed or is inhibited so that the target animal has the characteristics associated with the retinitis pigmentosa disease.
In a preferred embodiment of the present invention, the full-length sequence or partial sequence of the Ythdf1 gene in the genome of the target animal is not expressed or is inhibited by modification by one or a combination of mutation, deletion and insertion.
For example, such that the full length sequence, cDNA sequence or part of the exon sequence of the Ythdf1 gene is not expressed or is inhibited.
In a preferred embodiment of the present invention, the modification is performed by one or a combination of mutation, deletion and insertion such that the exon sequence or a part of the exon sequence of the Ythdf1 gene in the genome of the target animal is not expressed or is inhibited.
In a preferred embodiment of the present invention, at least one of the 1 st to 5 th exons of the Ythdf1 gene in the genome of the target animal is not expressed or is inhibited from being expressed by one or a combination of several of mutation, deletion and insertion.
In one embodiment, the 1 st, 1 st and 3 rd exons, 3 rd and 5 th exons sequences, 3 rd to 5 th exons sequences of the Ythdf1 gene are not expressed or are inhibited from expression.
In a preferred embodiment of the present invention, the 3 rd exon of the Ythdf1 gene in the genome of the target animal is not expressed or is inhibited by one or a combination of several of mutation, deletion and insertion.
In a preferred embodiment of the invention, the above construction method comprises modification of mutation, deletion or insertion by a combination of one or more of the following techniques:
gene knockout technology and gene editing technology.
In a preferred embodiment of the present invention, the gene knockout technology is Cre-loxP gene knockout technology.
In a preferred embodiment of the application of the present invention, the above-mentioned gene editing technology is selected from any one or a combination of several of CRISPR/Cas9 technology, ZFN technology and TALEN technology. It should be noted that, based on other gene editing techniques, it is within the scope of the present invention to make the target animal show the characteristics of the retinitis pigmentosa disease.
In a preferred embodiment of the application of the present invention, the target animal is a non-human mammal. In other embodiments, the non-human mammal is selected from any one of a mouse, a rat, a dog, a pig, a monkey, and an ape. One skilled in the art can adaptively select any non-human mammal as a target animal according to needs, and all the non-human mammals belong to the protection scope of the invention.
The target animal according to the present invention is not limited to the above-described animal, and may be any other type of animal such as rabbit, cow, horse, sheep, etc. Any animal can be selected as long as it is an animal having the Ythdf1 gene, and the Ythdf1 gene is knocked out from a rod cell of the animal to exhibit characteristics of a retinitis pigmentosa disease, and the animal is used as a retinitis pigmentosa disease model in the field of retinitis pigmentosa disease research, and belongs to the scope of the present invention.
In a preferred embodiment of the present invention, the above construction method is directed to the Ythdf1 gene in the genome of all cells of the target animal.
Further, in some embodiments of the present invention, the method for constructing a disease model specifically includes the following steps:
(1) Synthesis of a gRNA sequence SEQ ID NO:1 for replacing exon 3 of the Ythdf1 gene to be knocked out;
(2) The donor vector of the gRNA of the mouse Ythdf1 gene obtained in the step (1) and Cas9 mRNA are subjected to high-flux electrotransformation to obtain a mouse embryo stem cell with the Ythdf1 gene subjected to conditional knockout;
(3) Transplanting the fertilized eggs obtained in the step (2) into the uterus of a pseudopregnant mouse, delivering a offspring mouse, and sequencing and identifying to obtain a first-established mouse with Ythdf1 gene knocked out;
(4) Mating and breeding the chimeric mice obtained in the step (3) and wild mice, and screening heterozygote mice knocked out by Ythdf1 genes in offspring;
(5) Mating and breeding Ythdf1 gene knockout heterozygote mice obtained in the step (4) to obtain Ythdf1 gene knockout homozygote mice, and using the mice as a retinal pigment degeneration disease model.
The animal model obtained by the construction method has typical characteristics of the retinal pigment degeneration disease, has very wide application prospect, and can be used for researching the pathogenesis of the retinal pigment degeneration disease, for example, the animal model provides a basis for deeply knowing and researching 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 application of the retinal pigment degeneration disease model obtained by the construction method in screening medicines for preventing or treating retinal pigment degeneration diseases.
In a preferred embodiment of the application of the present invention, the application includes: administering a candidate drug to the model of a retinal pigment-modified disease, wherein the candidate drug is indicated as a drug for preventing or treating the retinal pigment-modified disease if the model of the retinal pigment-modified disease undergoes at least one of the following changes before and after administration of the candidate drug:
(1) After administration of the candidate drug, vision in the model of the retinitis pigmentosa disease is improved compared to before administration of the candidate drug; preferably a darkness vision improvement;
(2) After administration of the candidate drug, the outer retinal nuclear layer or inner retinal nuclear layer of the model of the retinitis pigmentosa disease is thickened compared to before administration of the candidate drug;
(3) Following administration of the candidate drug, the epiretinal node of the model of the retinitis pigmentosa disease increases as compared to prior to administration of the candidate drug.
The invention has the following beneficial effects:
the invention discovers for the first time that the Ythdf1 gene sequence in the genome of the target animal is not expressed or is inhibited to obtain a model of the retinal pigment degeneration disease, and the model shows the relevant characteristics of the retinal pigment degeneration disease. 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. Therefore, the novel model of the retinal pigment degeneration disease can be used in the fields of research of the retinal pigment degeneration disease and the like, and provides a richer model selection for research of the disease such as pathogenesis, mechanism and screening of related drugs. In addition, the development of the disease model provides a new target for the treatment or prevention of RP diseases.
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 is the construction and identification of a mouse with a specific knockout of Ythdf1 gene (KO) from a retinal rod cell;
fig. 2: detecting Ythdf1 gene knockout efficiency;
fig. 3: electroretinogram (ERG) detection results;
fig. 4: specific knockout of retinal rod cells Ythdf1 gene mouse retinal section immunohistochemical staining results;
fig. 5: specific knockout of retinal rod cells Ythdf1 gene mouse IHC staining result 1;
fig. 6: specific knock-out of retinal rod cells Ythdf1 gene mice IHC staining results 2.
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
The embodiment provides a construction method of a retinal pigment degeneration disease model, which takes a mouse as a target animal.
Ythdf1 gene knockout is mediated by Cre/loxP recombinase system, the knockout route is shown in FIG. 1A, and the specific operation is as follows:
1) Ythdf1 knockout mice were purchased from Siro Biotech Inc., taiku Chamber, jiangsu, su, taku, sha Xizhen Zhen Jixi Lu 69, (https:// www.cyagen.com/cn/zh-cn/sperm-bank-live/228994), and exon 3 of the model was deleted (FIG. 1A).
2) Mating and breeding the chimeric mice obtained in the step 1) and wild mice, and screening heterozygote mice knocked out by Ythdf1 genes from offspring;
3) Mating and breeding the Ythdf1 gene knockout heterozygote mice obtained in the step 2) to obtain Ythdf1 gene knockout homozygote mice, and the Ythdf1 gene knockout homozygote mice can be used as a retinal pigment degeneration disease model.
In this example, the offspring mice obtained in the above steps are subjected to genotyping, and the 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 PCR reaction system was configured as follows:
2×Taq Mix 10μL
tail tissue lysate 2. Mu.L
Primer 1 (Ythdf 1-loxP-Forward 1), 1. Mu.L (concentration: 10 mM)
Primer 2 (Ythdf 1-loxP-Forward 2), 1. Mu.L (concentration: 10 mM)
Primer 3 (Ythdf 1-loxP-Reverse), 1. Mu.L (concentration: 10 mM)
ddH 2 O 4μL。
The amplification primer sequences were as follows:
ythdf1-Forward1 sequence:
5’-CTGACTGGTCCTCTGTTGCTAGG-3’；
ythdf1-Forward2 sequence:
5’-CATGGAGATTGGGTACAGGCAGAC-3’；
ythdf1-Revers sequence:
5’-GTCCAAGAGACCCTGCATCACTG-3’；
the amplification procedure was as follows:
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 PCR product was taken in the wells and subjected to 120V constant pressure agarose electrophoresis for 15min.
FIG. 1B shows the results of Ythdf1 knockout mice, WT shows a wild-type control, and the band size is 1569bp; het represents a heterozygote with two bands 1569bp and 792bp; KO represents homozygote, and the band size is 792bp. According to the results shown in FIG. 1B, the PCR identification method provided in this example can effectively identify the genotype (heterozygous and homozygous) of the newborn mice for subsequent study.
Example 2
In this example, the gene knockout efficiency in the retinas of Ythdf1 knockout mice was analyzed by immunoblotting (Western blot) experiments.
The method comprises the following steps:
1) The control (Ythdf 1) +/+ ) And Ythdf1 knockout homozygote mice (Ythdf 1) -/- ) After sufficiently grinding, 200ul of protein lysate RIPA (Soy Biotechnology Co., ltd.) was added.
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 3 months of age, ytdf 1 was no longer expressed in the retinal cells of the knockout mice, YTHDF1 protein disappeared in retinal expression, indicating that it had been knocked out, and also demonstrated that example 1 successfully constructed Ytdf 1 knockout homozygote mice (Ytdf 1 -/ -)。
Example 3
This example shows that the Ythdf1 gene knockout homozygote mice constructed in example 1 at 8 months of age (Ythdf 1 -/- ) 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) Record displayAfter the wave signal is confirmed to be correct, the dark red light is turned off. It can be tried to record the dark adaptation light intensity of 0.003 cd/s.m 2 And (2) confirming 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 8 months, both a-wave and b-wave of the knockout mice were significantly reduced under dark adaptation conditions compared to wild-type mice, indicating that Ythdf1 resulted in impaired vision after rod cell knockout (fig. 3). FIG. 3A shows the electroretinogram waveforms of Ythdf1 wild type mice in dark adaptation and light adaptation at different light intensities; b is the electroretinogram waveform of Ythdf1 knockout mice under dark adaptation and light adaptation under different light intensities; C-E is a-wave and b-wave statistics for dark adaptation 3.0 and 10.0 and light adaptation 10.0, respectively, indicating that knockout mice have significantly reduced scotopic vision. The electroretinogram a wave and b wave have no obvious difference under the light condition, and the Ythdf1 does not influence the functions of the cone cells.
Example 4
This example the retinas of the knockout mouse model constructed in example 1 were subjected to paraffin section H & E staining.
Retinas of 8 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 8 months, compared with Ythdf1 +/+ (wild) mice, ythdf1 -/- The thickness of the outer and inner layers of the visual network was significantly thinner in (knockout) mice, indicating photoreceptor cell death (fig. 4). FIG. 4A shows Ythdf1 gene mouse retinal paraffin section H&E staining results, FIG. 4B is a graph showing statistics of thickness of outer nuclear layer of retina of Ythdf1 knockout mice at different sites, black line Ythdf1 +/+ (wild) mice, the red line (i.e., the lower line in FIG. 4B) is Ythdf1 -/- (knockout) mice.
Example 5
The present example performs an immunostaining experiment on frozen sections of retina.
The method specifically comprises the following steps: after the Ythdf1 gene knockout mice constructed in example 1 of 8 months old were sacrificed at the neck, eyeballs were quickly taken and put into 4% PFA, fixed on ice for 15min, cut on cornea, and then fixation on ice was 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 the graphShown at 5. At 8 months of age, the outer node antibodies Rhodopsin, PDE6B and CNGA1 were stained by frozen tissue sections of the retina, as compared to Ythdf1 +/+ (wild) mice, ythdf1 -/- The outer segments of the retinas of (knockdown) mice were significantly shortened and a significant degeneration characterization occurred.
In FIG. 5A, the inner and outer segments of rod cells are labeled with Rhodopsin and NaK antibodies, respectively, and the nuclei are counterstained with DAPI; FIG. 5B shows the counterstaining of nuclei with DAPI by labeling rod cell light transduction related proteins with PDE6B and CNGA1 antibodies, respectively.
Example 6
This example provides an immunostaining experiment on frozen sections of retina, with mice of 5 months of age in this application compared to example 5.
After the Ythdf1 gene knockout mice constructed in example 1 of 8 months old were sacrificed at the neck, eyeballs were quickly taken and put into 4% PFA, fixed on ice for 15min, cut on cornea, and then fixation on ice was 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 5 months of age, the neuroglia marker GFAP is stained by frozen tissue sections of the retina, and compared with a wild type mouse, the retina of the knockout mouse shows obvious gliosis and enhanced inflammatory response, which indicates retinal damage.
In summary, it can be seen that, in the embodiment of the invention, by taking a mouse as an example, the CRISPER/Cas9 knockout technology is used for knocking out the Ythdf1 gene, so that the mouse shows typical characterization of retinal pigment degeneration diseases such as visual impairment, shortening and degradation of extraocular nodes, loss of visual rod cells and the like. Thus, it is fully demonstrated that the knockout of the Ythdf1 gene can cause the target animal to exhibit retinal pigment degeneration disease. The Ythdf1 knockout animal can be used as a model of retinal pigment degeneration disease. The disease model can be used in the fields of research of retinal pigment degeneration diseases and the like, and provides a new model for research of the diseases such as pathogenesis, mechanism and screening of related medicines.
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 (5)
1. The construction method of the retinal pigment degeneration disease model is characterized by comprising the following steps: modification by deletion allows for the genome of all cells of a mouseYthdf1Exon 3 of the gene was not expressed to give Ythdf1 knockout homozygote mice.
2. The construction method according to claim 1, characterized in that it comprises the deletion modification by means of a combination of one or several of the following techniques:
gene knockout technology and gene editing technology; the gene knockout technology is Cre-loxP gene knockout technology, and the gene editing technology is selected from any one or a combination of a plurality of CRISPR/Cas9 technology, ZFN technology and TALEN technology.
3. Use of a model of a retinitis pigmentosa disease obtained by the construction method according to any one of claims 1-2 for screening a medicament for preventing or treating a retinitis pigmentosa disease.
4. The application according to claim 3, characterized in that it comprises: administering a candidate drug to the model of a retinal pigment-modified disease, wherein the candidate drug is indicated as a drug for preventing or treating a retinal pigment-modified disease if the model of a retinal pigment-modified disease changes at least one of the following before and after administration of the candidate drug:
(1) An improvement in vision of the model of the retinal pigment degeneration disease after administration of the drug candidate compared to before administration of the drug candidate;
(2) After administration of the candidate drug, the outer retinal nuclear layer or inner retinal layer of the model of the retinitis pigmentosa disease is thickened compared to before administration of the candidate drug;
(3) After administration of the candidate drug, the epiretinal node of the model of the retinitis pigmentosa disease increases as compared to before administration of the candidate drug.
5. The use according to claim 4, wherein the vision improvement of the model of retinitis pigmentosa disease is: the darkness vision of the model of the retinal pigment degeneration disease is improved.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111074309.1A CN113621649B (en) | 2021-09-14 | 2021-09-14 | Construction method and application of retinal pigment degeneration disease model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111074309.1A CN113621649B (en) | 2021-09-14 | 2021-09-14 | Construction method and application of retinal pigment degeneration disease model |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113621649A CN113621649A (en) | 2021-11-09 |
| CN113621649B true CN113621649B (en) | 2023-05-26 |
Family
ID=78390050
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111074309.1A Active CN113621649B (en) | 2021-09-14 | 2021-09-14 | Construction method and application of retinal pigment degeneration disease model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113621649B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114085837B (en) * | 2021-11-19 | 2024-04-12 | 中山大学 | Cell line for knocking out gene YTHDF1 and construction method thereof |
| CN114255823B (en) * | 2021-12-08 | 2024-09-13 | 四川省医学科学院·四川省人民医院 | Construction method and application of retina degenerative disease model |
| CN114752623B (en) * | 2022-01-06 | 2024-02-09 | 上海市第一人民医院 | Construction method and application of animal model of retinal pigment degeneration disease |
| CN114561408B (en) * | 2022-01-24 | 2023-04-21 | 四川省医学科学院·四川省人民医院 | Construction method and application of retinal pigment degeneration disease model |
| 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 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2020059719A (en) * | 2019-11-20 | 2020-04-16 | オックスフォード ユニヴァーシティ イノヴェーション リミテッド | Treatment of retinitis pigmentosa |
| CN111269945A (en) * | 2020-02-25 | 2020-06-12 | 四川省人民医院 | Method for constructing retinal pigment degeneration disease model by using Gm20541 gene and application |
| CN112715484A (en) * | 2020-12-29 | 2021-04-30 | 四川省人民医院 | Method for constructing retinal pigment degeneration disease model, application and breeding method |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102308212A (en) * | 2008-12-04 | 2012-01-04 | 加利福尼亚大学董事会 | Materials and methods for determining diagnosis and prognosis of prostate cancer |
| 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 |
| CN111500640B (en) * | 2020-04-17 | 2021-09-10 | 四川省人民医院 | Construction method and cultivation method of RP disease model |
-
2021
- 2021-09-14 CN CN202111074309.1A patent/CN113621649B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2020059719A (en) * | 2019-11-20 | 2020-04-16 | オックスフォード ユニヴァーシティ イノヴェーション リミテッド | Treatment of retinitis pigmentosa |
| CN111269945A (en) * | 2020-02-25 | 2020-06-12 | 四川省人民医院 | Method for constructing retinal pigment degeneration disease model by using Gm20541 gene and application |
| CN112715484A (en) * | 2020-12-29 | 2021-04-30 | 四川省人民医院 | Method for constructing retinal pigment degeneration disease model, application and breeding method |
Non-Patent Citations (1)
| Title |
|---|
| Mettl14-mediated m6A modification is essential for visual function and retinal photoreceptor survival;Yeming Yang等;BMC Biology;第20卷(第140期);第1-19页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113621649A (en) | 2021-11-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113621649B (en) | Construction method and application of retinal pigment degeneration disease model | |
| CN114561408B (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 | |
| Rachel et al. | Combining Cep290 and Mkks ciliopathy alleles in mice rescues sensory defects and restores ciliogenesis | |
| CN114916502B (en) | Construction method and application of retinal pigment degeneration disease model | |
| CN111269945B (en) | Method for constructing retinal pigment degeneration disease model by using Gm20541 gene and application | |
| Mansergh et al. | Mutation of the calcium channel gene Cacna1f disrupts calcium signaling, synaptic transmission and cellular organization in mouse retina | |
| CN115176760B (en) | Method for constructing retinal pigment degeneration disease model, application and breeding method | |
| CN109182355B (en) | The construction method of retinal neovascularization disease model and application | |
| US20200375157A1 (en) | Construction method for animal model for retinitis pigmentosa diseases and application | |
| CN111500640B (en) | Construction method and cultivation method of RP disease model | |
| Sajedi et al. | Analysis of mouse models carrying the I26T and R160C substitutions in the transcriptional repressor HESX1 as models for septo-optic dysplasia and hypopituitarism | |
| CN111778281B (en) | Construction method and application of retina bipolar cytopathy model | |
| US11653636B2 (en) | Method of making a rat model of retinal degeneration and rat model made thereby | |
| CN114255823B (en) | Construction method and application of retina degenerative disease model | |
| EP4061823A1 (en) | Genetically-directed sparse and complete labeling of brain cells | |
| CN117296799B (en) | Construction method and application of retinal pigment degeneration disease model | |
| CN117230077B (en) | Application of Hakai gene in RP disease model construction and construction method | |
| EP1619248B1 (en) | Mouse with deficiency of glutamate transporter glast function | |
| Pandey et al. | Advances in Genetic Diagnosis and Treatment of Hearing Loss—A Thirst for Revolution | |
| KR100430199B1 (en) | Transgenic mouse with disrupted calcium ion channel alpha1D gene and production method thereof | |
| CN109090040B (en) | A kind of Wnt10aflox/floxThe construction method of mouse model | |
| CN117305364A (en) | Method for constructing retina degeneration disease model by using gene manipulation technology and application | |
| CN115777626A (en) | Construction method and application of Ift140 gene rod-like cell conditional knockout mouse model | |
| JP2006158397A (en) | F066 transgenic mouse expressing pathogenic variant of sca2 gene |
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 |