CN113308476B - 一种FcRn基因人源化动物模型的构建方法 - Google Patents
一种FcRn基因人源化动物模型的构建方法 Download PDFInfo
- Publication number
- CN113308476B CN113308476B CN202110517875.9A CN202110517875A CN113308476B CN 113308476 B CN113308476 B CN 113308476B CN 202110517875 A CN202110517875 A CN 202110517875A CN 113308476 B CN113308476 B CN 113308476B
- Authority
- CN
- China
- Prior art keywords
- mouse
- fcrn
- humanized
- fcrn gene
- human
- 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
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 title claims abstract description 56
- 238000010171 animal model Methods 0.000 title claims abstract description 19
- 238000010276 construction Methods 0.000 title abstract description 9
- 241001465754 Metazoa Species 0.000 claims abstract description 6
- 230000001225 therapeutic effect Effects 0.000 claims abstract description 5
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 32
- 238000000034 method Methods 0.000 claims description 20
- 239000013598 vector Substances 0.000 claims description 20
- 238000011577 humanized mouse model Methods 0.000 claims description 13
- 241001529936 Murinae Species 0.000 claims description 11
- 108091033409 CRISPR Proteins 0.000 claims description 10
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 10
- 239000013612 plasmid Substances 0.000 claims description 7
- 201000010099 disease Diseases 0.000 claims description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000012163 sequencing technique Methods 0.000 claims description 6
- 238000013518 transcription Methods 0.000 claims description 6
- 230000035897 transcription Effects 0.000 claims description 6
- 238000010172 mouse model Methods 0.000 claims description 5
- 238000012408 PCR amplification Methods 0.000 claims description 4
- 238000005516 engineering process Methods 0.000 claims description 3
- 238000000338 in vitro Methods 0.000 claims description 3
- 238000010354 CRISPR gene editing Methods 0.000 claims description 2
- 210000000805 cytoplasm Anatomy 0.000 claims description 2
- 108020004999 messenger RNA Proteins 0.000 claims description 2
- 108091006020 Fc-tagged proteins Proteins 0.000 abstract description 11
- 239000003814 drug Substances 0.000 abstract description 10
- 229940079593 drug Drugs 0.000 abstract description 8
- 210000002966 serum Anatomy 0.000 abstract description 8
- 238000012360 testing method Methods 0.000 abstract description 2
- 238000011282 treatment Methods 0.000 abstract description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 33
- 241000699670 Mus sp. Species 0.000 description 32
- 108090000623 proteins and genes Proteins 0.000 description 28
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 27
- 108020004414 DNA Proteins 0.000 description 23
- 102000053602 DNA Human genes 0.000 description 23
- 102000004169 proteins and genes Human genes 0.000 description 19
- 108010068617 neonatal Fc receptor Proteins 0.000 description 13
- 238000001962 electrophoresis Methods 0.000 description 10
- 230000008685 targeting Effects 0.000 description 10
- 230000003834 intracellular effect Effects 0.000 description 8
- 238000011740 C57BL/6 mouse Methods 0.000 description 6
- 102000007562 Serum Albumin Human genes 0.000 description 6
- 108010071390 Serum Albumin Proteins 0.000 description 6
- 239000006180 TBST buffer Substances 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 108091026890 Coding region Proteins 0.000 description 5
- 101150050927 Fcgrt gene Proteins 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 230000027455 binding Effects 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000010200 validation analysis Methods 0.000 description 4
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102000004243 Tubulin Human genes 0.000 description 3
- 108090000704 Tubulin Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 229940125644 antibody drug Drugs 0.000 description 3
- 238000010241 blood sampling Methods 0.000 description 3
- 235000013601 eggs Nutrition 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000031146 intracellular signal transduction Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 108020005345 3' Untranslated Regions Proteins 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 108010087819 Fc receptors Proteins 0.000 description 2
- 102000009109 Fc receptors Human genes 0.000 description 2
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 2
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 2
- 241000581650 Ivesia Species 0.000 description 2
- 239000002033 PVDF binder Substances 0.000 description 2
- 102100022873 Ras-related protein Rab-11A Human genes 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 210000002459 blastocyst Anatomy 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000012239 gene modification Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 102000013446 GTP Phosphohydrolases Human genes 0.000 description 1
- 108091006109 GTPases Proteins 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 101100066437 Homo sapiens FCGRT gene Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101100425749 Mus musculus Tnfrsf18 gene Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 101150060955 RAB11A gene Proteins 0.000 description 1
- 102000028589 Rab4 Human genes 0.000 description 1
- 108050007312 Rab4 Proteins 0.000 description 1
- 101710136851 Ras-related protein Rab-11A Proteins 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 206010052779 Transplant rejections Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000000611 antibody drug conjugate Substances 0.000 description 1
- 238000011230 antibody-based therapy Methods 0.000 description 1
- 229940049595 antibody-drug conjugate Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 210000004082 barrier epithelial cell Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002121 endocytic effect Effects 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 230000008497 endothelial barrier function Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 230000004890 epithelial barrier function Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 230000028023 exocytosis Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000007185 extracellular pathway Effects 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 238000011866 long-term treatment Methods 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000001839 systemic circulation Effects 0.000 description 1
- 229940126622 therapeutic monoclonal antibody Drugs 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
Images
Classifications
-
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
-
- 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
- A01K67/0278—Knock-in vertebrates, e.g. humanised 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
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
-
- 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
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- 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
- A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
-
- 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
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Environmental Sciences (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
- Endocrinology (AREA)
- Pathology (AREA)
- Rheumatology (AREA)
- Urology & Nephrology (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明提供了一种FcRn基因人源化动物模型的构建方法,该人源化动物可用于研究人Fc融合蛋白的药物PK,也可用于不同抗体以及同一抗体不同突变体之间的半衰期的差异评价,或评价人Fc融合蛋白最大半衰期数据,预估达到治疗性血清浓度的最小剂量,从而减少了临床试验期间对潜在危险的剂量递增治疗的需求。
Description
技术领域
本发明属于动物基因工程和基因遗传修饰领域,具体涉及基于CRISPR/Cas9技术的FcRn基因修饰人源化动物模型的构建方法及其在生物医药的应用。
背景技术
治疗性单克隆抗体(mAb)已成为一类重要的药物,美国FDA批准了40多种基于抗体的疗法,涵盖一系列疾病,包括癌症,自身免疫性疾病,传染病,神经退行性疾病,黄斑变性,骨质疏松症和移植排斥,并且在临床试验中的应用更多。自1975年杂交瘤技术和单克隆抗体(mAb)开发突破以来,抗体工程学的学者们已经将mAb从第一代高度免疫原性鼠类和嵌合抗体,迅速发展到耐受性更好的人源化和完全人源mAb。最近,各种抗体样形式进一步发展,包括Fc融合蛋白、抗体-药物偶联物和双特异性抗体等。除了出色的特异性和靶向疗效外,mAbs还拥有较长的药代动力学(PK)半衰期。另外由于mAb在全身循环中清除率较低,这使得采用mAbs的给药频率相比于其它肽或小分子对应药物的给药频率低的多,更加适用于慢性疾病的长期治疗。
mAb的PK特性取决于其大小尺寸、相对极性、Fc受体结合以及与靶抗原的特异性结合。其中Fc受体结合由细胞表面FcRn决定。研究表明,FcRn能够结合血清中的IgG和血清白蛋白,将其胞吞进入细胞内“保护起来”,再通过胞吐的形式释放他们,从而达到IgG和血清白蛋白延长的血清半衰期以及跨越内皮和上皮屏障的运输,增加IgG和血清白蛋白的总体生物利用度。
mAb的临床前测试通常在啮齿动物和非人类灵长类动物(NHP)中进行,以了解其功效和给药前有毒。通常,小鼠是用于疾病建模和临床前药代动力学(PK)分析的公认的和优选的体内验证模型。科学家们通过小鼠进行体内验证,包括给药抗体的吸收、分布、代谢和排泄等。然而,由于人鼠种间的差异,传统的近交系或远交系小鼠已逐渐被证明不完全适用于研究人Fc融合蛋白的PK特征。
人鼠FcRn在基因及蛋白上存在固有的种间差异,基于小鼠评价人类mAb抗体药物时,由于小鼠的FcRn对人类IgG的Fc段亲和力不同,其不能完全模拟临床上的PK和利用率。基于这一问题,有学者使用转基因方式(Transgene,TG)将human FCRN完整的基因编码区导入小鼠基因组内,获得的人源化FcRn小鼠在美国的Jackson实验室已提供商用。该小鼠目前已被广泛利用与人类IgG抗体药物PK研究,但是该模型在检测人类IgG抗体药物PK和最大半衰期数据数据上通常与临床数据存在偏差。
FcRn蛋白为跨膜蛋白,其胞外区(包括a1、a2和a3亚基)与B2m蛋白组成类似MHCI空间结构的蛋白复合体,负责与IgG的Fc段结合。FcRn蛋白的胞内区与Rab4和Rab11或仅与Rab11结合。Rab蛋白是类Ras样GTPases,已知Rab蛋白在胞吞和胞外途径中均起调节功能。目前商用的人源化FcRn小鼠,其FcRn蛋白胞内和胞外区序列完全为人类序列,胞外区负责与人源Fc结合,但是其胞内区与鼠源胞内信号传导是否存在种间差异性,尚未可知。
我们开发了一种表达人源化FcRn的小鼠模型,该模型采用原位Knockin的方式,将小鼠FcRn基因的胞外区替换为人源序列,而跨膜及胞内区则保留完整的鼠源序列。同时保留了小鼠FcRn基因的启动子、UTR等调控区。在构建过程中,我们优化了制备稳定表达人源化FcRn基因的小鼠模型的具体操作方法,该方法探索了适合插入小鼠FcRn基因的人FcRn基因区段,确保在成功表达人源化FcRn基因的基础上不影响小鼠其他功能,此过程中还优化了表达人源化FcRn的整合载体上的元件构成、敲除鼠源基因的最佳sgRNA、以及鉴定筛选所使用的引物和酶切方案等,确保了制备人源化FcRn动物模型的高效性。
本发明获得的人源化小鼠在模拟人FcRn与人IgG Fc亲和力的同时,更能够模拟小鼠体内FcRn的表达丰度及胞内信号传导。该人源化小鼠用于研究人Fc融合蛋白的药物PK,解决评价抗体半衰期存在临床差异的问题。该小鼠也可用于不同抗体以及同一抗体不同突变体之间的半衰期的差异评价,增加抗体临床前药物评价内容。另外,该人源化小鼠可用于评价人Fc融合蛋白最大半衰期数据,预估达到治疗性血清浓度的最小剂量,从而减少了临床试验期间对潜在危险的剂量递增治疗的需求。
发明内容
本发明提供了一种人源化FcRn基因的DNA序列,其特征在于,该DNA序列中包含来自人源FcRn基因的部分Exon2、Intron2、Exon3、Intron3、Exon4、Exon5、Intron5、部分Exon6,以及来自非人动物源FcRn基因的exon1、intron1、部分exon2、部分exon6、intron6、exon7,所述DNA序列如SEQ ID NO:1所示。
本发明还提供了包含所述DNA序列的载体。
优选地,所述载体包含非人动物源5’UTR编码区及同源臂序列、人源FcRn基因、非人动物源FcRn基因、非人动物源3’UTR及同源臂序列。
本发明还提供所述DNA序列或所述载体在制备人源化FcRn基因动物模型中的用途。
本发明还提供一种制备FcRn基因人源化动物模型的方法,其特征在于,包括以下步骤:
(1)构建表达针对非人动物源FcRn基因的sgRNA的质粒;
(2)将人源FcRn基因的DNA序列、非人动物源FcRn基因的3’同源臂和5’同源臂连接至质粒上,构建人源化FcRn基因的载体;
(3)将步骤(1)所述质粒体外转录获得的sgRNA与步骤(2)的载体以及Cas9 mRNA或Cas9蛋白注射至鼠受精卵细胞质或细胞核中,移植入受体动物母体生产FcRn基因人源化非人动物模型;
所述载体为如前所述的载体。
优选地,针对非人动物源SgRNA基因的sgRNA的序列如SEQ ID NO:2和SEQ ID NO:3所示。
优选地,所述方法进一步包括利用PCR扩增引物鉴定所述动物模型的步骤。
优选地,所述方法包括利用下表所示PCR扩增引物鉴定人源化非人动物基因型的步骤:
其中KI为中靶基因型;WT为野生型;KI/KI为纯合子;KI/WT为杂合子。
优选地,所述方法进一步包括利用测序引物鉴定所述动物模型的步骤。
优选地,其中所述测序引物如下所示:
本发明还提供如前所述的DNA序列或者所述的载体或所述的方法在制备模拟人FcRn与人IgG Fc亲和力的动物模型,或制备模拟小鼠体内FcRn的表达丰度及胞内信号传导的动物模型,或制备用于研究人Fc融合蛋白的药物PK以解决评价抗体半衰期存在临床差异的问题的动物模型,或制备用于不同抗体以及同一抗体不同突变体之间的半衰期的差异评价的动物模型,或制备评价人Fc融合蛋白最大半衰期数据的动物模型中的应用,所述应用为非疾病诊断或非疾病治疗目的。
所述Fc融合蛋白是指利用基因工程等技术将某种具有生物活性的功能化学分子与免疫球蛋白(IgG、IgA等)的Fc片段融合而产生的新型重组蛋白。其不仅保留了功能蛋白分子的生物学活性,还具有一些抗体的性质。
本发明还提供一种特异性靶向非人动物FcRn基因的sgRNA,其序列如SEQ ID NO:2和SEQID NO:3所示。
优选地,所述非人动物为小鼠。
本发明具有以下积极效果:
1、本发明利用基因修饰的方法构建FcRn部分人源化小鼠,将小鼠FcRn基因的胞外区替换为人源序列,而跨膜及胞内区则保留完整的鼠源序列。制作成功的人源化模型拥有人源胞外区,可模拟人体内FCRN对人类IgG的Fc段亲和力,而鼠源胞内区则保证其胞内信号传导不受影响,将外部刺激忠实地转化为胞内行为。
2、本发明构建的人源化小鼠与普通小鼠相比,采用原位Knockin方式实现了FcRn的胞外区人源化,并且保留了完整的鼠源跨膜及胞内信号传导区域,同时使小鼠本身FcRn胞外区缺失,排除鼠源FcRn干扰数据。
3、本发明构建的人源化小鼠保留了小鼠FcRn基因的启动子、UTR等调控区,其FcRn在体内不同器官组织的表达情况更接近于小鼠本身。该小鼠在模拟人FcRn与人IgG Fc亲和力的同时,更能够模拟小鼠体内不同组织器官FcRn的表达丰度,可用于不同组织的给药的药物PK变化分析。
4、该人源化小鼠可用于研究人Fc融合蛋白的药物PK,解决评价抗体半衰期存在临床差异的问题。该小鼠也可用于不同抗体以及同一抗体不同突变体之间的半衰期的差异评价,增加抗体临床前药物评价内容。另外,该人源化小鼠可用于评价人Fc融合蛋白最大半衰期数据,预估达到治疗性血清浓度的最小剂量,从而减少了临床试验期间对潜在危险的剂量递增治疗的需求。
5、本发明提供了优化的制备稳定表达人源化FcRn基因的具体操作方法,我们对该方法中所使用的人源化区域的选择、同源重组过程中的同源臂的设计、打靶载体的构建、靶向鼠源FcRn的最佳sgRNA、以及鉴定筛选所使用的引物和酶切方案等都进行了大量的筛选和优化,确保了制备目的动物模型的高效制备。
附图说明
图1为F0小鼠PCR鉴定琼脂糖电泳图。
图2为F1小鼠PCR鉴定琼脂糖电泳图。
图3为肝组织中hFcRn/mFcRn蛋白表达。
图4为人源的IgG1亚型抗体在C57BL/6-hFcRn小鼠体内半衰期的检测。
具体实施方式
下面结合附图对本发明做进一步的详细说明,以令本领域技术人员参照说明书文字能够据以实施。
实施例1:FcRn人源化打靶同源DNA供体构建
利用同源重组将人源FCRN基因的编码蛋白膜外区替换C57BL/6鼠源FcRn基因的蛋白胞外区编码区,保留C57BL/6小鼠的UTR序列,构建C57BL/6背景的打靶载体包含SEQ IDNO:1所示序列:其中1-782bp为C57BL/6鼠源5’UTR编码区及同源臂序列,依次包含exon1、intron1、部分exon2;783-2838bp为人源FCRN基因部分,依次包含部分Exon2、Intron2、Exon3、Intron3、Exon4、Exon5、Intron5、部分Exon6的序列;2839-3727bp部分为C57BL/6鼠源FcRn区、3’UTR及同源臂序列,依次包含部分exon 6、intron6、exon7。
SEQ ID NO:1
ggctggacagaggcccaaggggtgtgcttaggagctagtgggtggagttggatgccctcagagttctccagtcctaactgtgtacagacaggatgtaagagaagaactggaggctctaagcagaggatccatcggctgcaggcagagggaagagggcctctgtgaggaacaggctgagcgtcagaggaggaggcccaggcctggttctctaggtatgagggaccagcaccccaagaggaaagagggttggggcctggagctccaggtctgagggaggagtgttatgacctgagggggagagaggaggctggggtctgaggaaggagagcaggaccccgggcacctgagtctgagggatgaagccggagcctagggttttgggtttgaggaaagatcttggaactggactcctggatctgagggaggtggaactggactcctggcttgggtttgagggctcctgaggctgaaatctgagcccctgggaatgagggaggaggagaaggcctggatcctctggtctgagggaggagaaaaaggaaataacagaagtctgagacaaattttggtctgtctagctctgtaattaattaactaaagtggatcaaatgagaaggtgaaagttcacagaggaacactcctgtctgtcgtcttggactgggtctccatcccaccatccagcgtcctggtctacgaagagtccacagggaccttgtgaagaatcaacaaggcggggtccagaggagtcacgtgtcccttccactccgggtcaccctgtcggaatgggggtcccgcggcctcagccctgggcgctggggctcctgctctttctccttcctgggagcctgggcgcaggtgagggccgctccgggccagggccctgctgcaggcgggcggcgggaggcggccccaggcaggcaggggcgaagccagcgggacccgagttccccgcgagcccctggcgctgccttctccgctcaattaactttctcaacgtctcctgctgggcctgaggctgggaaccacctgtcgcctcttgtgtctgtttccctctctctctctgggtctctgtccccctctctttctgggtctcttgtctctctctctctgggtctctgtccccccccccccgggtttctgtcccctctctctgaatctgtccccctccctccataatagattcttctccctccctgggtatctgtcccactgcagtctagttccccgcccgtgtgctcccttcagctctgtttctgtctgcagaaagccacctctccctcctgtaccaccttaccgcggtgtcctcgcctgccccggggactcctgccttctgggtgtccggctggctgggcccgcagcagtacctgagctacaatagcctgcggggcgaggcggagccctgtggagcttgggtctgggaaaaccaggtgtcctggtattgggagaaagagaccacagatctgaggatcaaggagaagctctttctggaagctttcaaagctttggggggaaaaggtgagattccggtctggaggggcaaggggccgggtccatgctccggggccccgcttacctgtgtttgggcgccccaggtccctacactctgcagggcctgctgggctgtgaactgggccctgacaacacctcggtgcccaccgccaagttcgccctgaacggcgaggagttcatgaatttcgacctcaagcagggcacctggggtggggactggcccgaggccctggctatcagtcagcggtggcagcagcaggacaaggcggccaacaaggagctcaccttcctgctattctcctgcccgcaccgcctgcgggagcacctggagaggggccgcggaaacctggagtggaaggagcccccctccatgcgcctgaaggcccgacccagcagccctggcttttccgtgcttacctgcagcgccttctccttctaccctccggagctgcaacttcggttcctgcggaatgggctggccgctggcaccggccagggtgacttcggccccaacagtgacggatccttccacgcctcgtcgtcactaacagtcaaaagtggcgatgagcaccactactgctgcattgtgcagcacgcggggctggcgcagcccctcagggtggagctgggtgaggtcccgccaggtggtgatgctcctggtttcccgttgccttgtctcactgctgcgccggtccttcctgagtctgaccttcctccccactgctgccacctccttgaatctgactgccttgaacctcacgcctgtcagtgcccccaaaacctgatggcttgtccttcccaaggccaactgccttccgtctcctgctgcttctggcctcactgagtctgaagagctgttaactaccatggccagtcctccctgagtctgaccatcttccatcctgctgctgctgctgctgctgctgcgggtcttcctggaatctgaccattcgttgtctgctatgcccgtcctcaccaagactgactgcctgctgctttgctactgcccgggcccatgagactgacttcccactgctctgcctgcctctccccactgcactggcacagccccgccttgccgctgctgatccattgccggtgtgaccgcggccgctcgtgctgtagctggttcttacgtccaacctgggggcagtctgcccagatcgcctgcctggcctcgcctctgcccatcagacacttggtgctggaatctccgaggctgggagggactgggagcgccccagttctgtgtcctgactgggtggggtggaggggctgctccacatctcacagcgttctctggctgcagaatctccagccaagtcctccgtgcctgtggttggaatcgttcttggtttattgctggtggtagtggccatcgcaggcggtgtgctgttgtggggcaggatgcgcagcggtctgccaggtatatgcaggatgggggaggggcggggccgtgtgcaggattgggtgggagtggccttgtgcaggatgagggaggggcggggccgtgtgcaggattggggtggggcgtggccgtgtgcagaatgagggaggggcgtggccgtgtgcaggattggggaggggagggaaggggccgtgtgcaggattggggaggggcgtagctgtgtgcaggattggggaagggaggggccgtgtgcaggattgggggtcgtggcaggaagtagcgactcagaaagaaagaggggaggcagagagataatagagacaaaagagtccctgaccaccctgggaaaagagatgggaacagagaccatagcgtgaccctgtcaggtgtgttgggtgggtgagaccttagatacttgtgctgacctctgttcctctctctcagccccatggctttctctcagcggcgatgactctggtgacctgttgcctggtgggaacttgcccccagaagctgaacctcaaggtgcaaatgcctttccagccacttcctgatgcagactcgggccccctgcccactgcagcctttcgggctgtgtgacctcctgaactgtctccgagcctcctgagggagcctgggcccgatgtcctcccatggatccctgcttttgtggcctgcttcagtttcccttcttaatgtacatggttgttttccatctccacataaatttggccccaaatctgtgtgtgcatcgttattctcaagtttcaagcagctggaataaattgaacgcgtctgggaaagatcata
实施例2FcRn人源化小鼠sgRNA设计
根据替换片段确定sgRNA大概区域。使用sgRNA测评软件,针对目的片段选取脱靶率最低几组序列,如下序列作为待选sgRNA。设计并合成识别5’端靶位点和3’端靶位点,并构建sgRNA表达载体。两端sgRNA识别位点分别位于小鼠Tnfrsf18基因的第一个和第五个外显子上。
sgRNA转录制备方法:以PrimerStar或PrimerStar Max体系,sgRNA-F、sgRNA-R为引物,测序正确的puc57-sgRNA质粒(1:30稀释)为模板进行PCR,PCR产物进行纯化,制备sgRNA转录制备模板。使用T7-ShortScript体外转录试剂盒(AM1354)进行sgRNA的转录。
sgRNA筛选:将待选sgRNA与Cas9蛋白进行孵育后,将混合液注射至0.5天的受精卵中,培养至囊胚阶段后,进行小鼠FcRn基因的KO阳性率的鉴定,以此筛选切割活性高的sgRNA。
sgRNA切割鉴定方法:对收集的囊胚进行PCR扩增,根据PCR结果进行统计(发生KO才可扩增出目的条带),统计发生KO的概率,根据各sgRNA所处位置和各种组合切割效率最终选择表1所示的sgRNA进行后续实验,该对sgRNA的切割效率高达85%,且其所处位置与其他sgRNA相比更加有利于基因重组,故而择之。
表1
sgRNA名称 | 序列 | PAM |
gRNA1 | AGTGGCATCCCCATTCCGAC | AGG |
gRNA2 | AGCCAGATCTTCTGTGCCTG | TGG |
实施例3FcRn人源化小鼠模型建立
将实施例2设计得到的sgRNA以及实施例1设计的FcRn人源化打靶同源DNA供体,Cas9蛋白混合后,注射到0.5天的小鼠受精卵中,移植至0.5天假孕雌鼠体内,等小鼠出生后,经基因鉴定筛选出中靶小鼠(F0)。
人源化小鼠F0代基因型鉴定:对获得的F0小鼠的鼠尾基因组DNA分别使用两对引物进行中靶后的两端PCR鉴定,引物710181-FCRN-5tF1/710181-hFCRN-5tR1分别位于5’同源臂外及打靶载体的人源片段内,如该对引物扩增产生PCR产物,说明目标载体在小鼠基因组5’进行了有效插入;710181-hFCRN-3tF1/710181-FCRN-3tR1分别位于打靶载体的人源片段内及3’同源臂外,如该对引物扩增产生PCR产物,说明目标载体在小鼠基因组3’进行了有效插入。
表2 F0及F1鉴定FcRn正确插入引物
注:KI为中靶基因型;WT为野生型;KI/KI为纯合子;KI/WT为杂合子
PCR反应体系以及反应条件如表3和4所示:
表3PCR反应体系
试剂(Vazyme P112-03) | 体积(μl) | 规格 |
2×Taq Master Mix,Dye Plus | 12.5 | \ |
ddH2O | 9.5 | \ |
Primer A(10pmol/μl) | 1 | 10μM |
Primer B(10pmol/μl) | 1 | 10μM |
Template | 1 |
表4PCR反应条件
对两端PCR鉴定阳性的小鼠克隆进行测序验证,打靶载体在小鼠基因组中替换编码区后,测序正确的克隆经鉴定为阳性F0小鼠。
表5测序引物
结果:
1.F0鉴定结果:
基于C57BL6/JGpt小鼠背景打靶获得4只阳性F0小鼠。hFcRn F0鼠尾DNA鉴定结果如图1所示(5’arm标记为hFcRn-KI-target 5’端鉴定电泳图,3’arm标记为hFcRn-KI-target3’端鉴定电泳图,WT对照为C57BL6/JGpt基因组DNA;N为空白对照,无模板的对照;P为阳性对照;M为TRANS 2K PLUS II条带:8000bp\5000bp\3000bp\2000bp\1000bp\750bp\500bp\250bp\100bp)。其中1#,13#,18#,30#小鼠的人源FCRN基因5’及3’鉴定及测序均为阳性,表明这些小鼠为正确进行基因重组的F0阳性小鼠。
2.F1鉴定结果:
C57BL6/JGpt背景的F0阳性小鼠与C57BL6J/Gpt背景鼠配繁获得F1代,对F1代鼠尾进行hFcRn基因鉴定,F1代小鼠PCR实验结果见图2(从上至下依次为hFcRn-KI-target 5’端、3’端及WT鉴定电泳图,WT为野生鼠基因组DNA做模板;N为空白对照,无模板的对照;P为阳性对照;TRANS 2K PLUS II条带:8000bp\5000bp\3000bp\2000bp\1000bp\750bp\500bp\250bp\100bp)。37#,43#,44#,46#小鼠的人源化hFcRn基因5’及3’鉴定均为阳性,表明小鼠为正确进行基因重组的阳性小鼠。F1大量扩繁后进行互配,获得FcRn人源化小鼠纯合子小鼠,简称C57BL/6-hFcRn。
实施例4C57BL/6-hFcRn小鼠中人源FCRN蛋白表达验证
通过Western Blot分析C57BL/6-hFcRn小鼠能够成功表达人源化蛋白。
FcRn蛋白广泛表达于内皮细胞,巨噬细胞,单核细胞衍生的树突细胞。我们采集C57BL/6-hFcRn小鼠及对照背景鼠的肝脏,检测组织中FcRn蛋白的表达。
FcRn蛋白Western Blot检测方法:
a)取材:对C57BL/6-hFcRn杂合小鼠、纯合小鼠和C57BL/6背景鼠(见小鼠信息列表)剪取肝脏。
表6待检小鼠样品信息列表
b)总蛋白提取及浓度测定:
加入预冷的RIPA蛋白裂解液(含蛋白酶抑制剂),将冻存组织置于其中。用匀浆器匀浆至无肉眼可见的絮状物,将样品放置于湿冰上30min,以使样品充分裂解。
4℃,12,000rpm离心30分钟,取上清至1.5mL EP管中,可分装或者保存于-80℃冰箱。提取的样品测定总蛋白浓度,采用BCA蛋白浓度测定试剂盒,实验操作按照试剂盒说明书进行。
c)SDS-PAGE电泳:
配制10%的分离胶与5%的浓缩胶。
上样前采用移液枪将上样孔内未凝固的胶吹洗干净。
上样,根据BCA蛋白定量所计算的上样体积进行蛋白样品上样,单孔上样量总蛋白为30ug。
电泳,恒压100V,待溴酚蓝跑到距离最底部约1厘米处即可终止电泳。
d)转膜
准备4张加厚滤纸和1张PVDF膜,膜要先在冷甲醇中浸泡1分钟,然后将滤纸和膜置于1×转膜液中平衡。按三明治结构将胶、PVDF膜放好,胶与膜之间避免产生气泡。放好之后将电泳槽置于冰水中,转膜过程中保持低温。
恒流350mA,hFcRn/mFcRn蛋白与内参蛋白共转膜90min。
e)封闭
含5%non-fat milk的1×TBST中室温封闭60min。
f)孵育一抗
α-Tubulin一抗采用含5%BSA的TBST的封闭液1:10000稀释;
GAPDH一抗采用含5%BSA的TBST的封闭液1:10000稀释;
hFcRn一抗采用含5%BSA的TBST的封闭液1:500稀释;
mFcRn一抗采用含5%BSA的TBST的封闭液1:400稀释;
4℃冷库振荡过夜。
g)洗膜,1×TBST洗膜三次,每次10min
h)孵育二抗
Goat anti-Rabbit HRP二抗1:10000稀释(对应一抗α-Tubulin、GAPDH、Anti-hFcRn);
Rabbit anti-Goat HRP二抗1:10000稀释(对应一抗Anti-mFcRn)
室温孵育2h。
i)洗膜,化学发光成像仪拍照
1×TBST洗膜三次,每次10min
ECL发光液,α-Tubulin/GAPDH蛋白曝光2s,hFcRn蛋白曝光60s,mFcRn蛋白曝光120s。
结果:
C57BL/6-hFcRn小鼠能够表达人源化的hFcRn蛋白。
实施例5C57BL/6-hFcRn小鼠可用于评价人源抗体半衰期的验证
将人源的IgG1亚型抗体分别注射到C57BL/6-hFcRn小鼠和C57BL/6JGpt小鼠腹腔中,通过不同时间点采集小鼠血液,分析其中人源的IgG1亚型抗体的浓度。
选用两种人源抗体human IgG1 a和human IgG1 b,两次实验结果均证实在C57BL/6-hFcRn小鼠中,人源的IgG1亚型抗体在体内的半衰期得到明显的延长。该数据显示C57BL/6-hFcRn小鼠可用于评价人源抗体半衰期。
人源抗体半衰期验证方法:
a)药物配制或稀释方法:
给药剂量:人源的IgG1亚型的抗体:30mg/kg。
给药体积:10mL/kg。
药物浓度:3mg/mL
给药方式:皮下注射
b)小鼠分组及采血方案
C57BL/6-hFcRn 6只(3♂+3♀),25g左右。
C57BL/6JGpt 6只(3♂+3♀),25g左右。
动物编号采血时间点:0h,5h,day2,day 4,day 7,每个点采集2只动物(1♂+1♀)。
(小鼠无法承受短时间多次血液采集)
表7小鼠分组及采血时间点
每个点采集100μL左右全血,分离血清:血液室温(22-25℃)放置30-60min,待血液标本自发完全凝集,4000rpm,2-4℃离心10min,收集血清标本,-20℃保存待测。c)检测蛋白在血清中的人IgG1浓度。
采用商品化人IgG1 ELISA检测试剂盒,实验操作按照试剂盒说明书进行。
结果:
与C57BL/6JGpt对比,两种人源抗体human IgG1 a和human IgG1 b在C57BL/6-hFcRn小鼠体内的半衰期得到明显的延长。
尽管本发明的实施方案已公开如上,但其并不仅仅限于说明书和实施方式中所列运用。它完全可以被适用于各种适合本发明的领域。对于熟悉本领域的人员而言,可容易地实现另外的修改。因此在不背离权利要求及等同范围所限定的一般概念下,本发明并不限于特定的细节和这里示出与描述的图例。
序列表
<110> 广东药康生物科技有限公司
<120> 一种FcRn基因人源化动物模型的构建方法
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3727
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggctggacag aggcccaagg ggtgtgctta ggagctagtg ggtggagttg gatgccctca 60
gagttctcca gtcctaactg tgtacagaca ggatgtaaga gaagaactgg aggctctaag 120
cagaggatcc atcggctgca ggcagaggga agagggcctc tgtgaggaac aggctgagcg 180
tcagaggagg aggcccaggc ctggttctct aggtatgagg gaccagcacc ccaagaggaa 240
agagggttgg ggcctggagc tccaggtctg agggaggagt gttatgacct gagggggaga 300
gaggaggctg gggtctgagg aaggagagca ggaccccggg cacctgagtc tgagggatga 360
agccggagcc tagggttttg ggtttgagga aagatcttgg aactggactc ctggatctga 420
gggaggtgga actggactcc tggcttgggt ttgagggctc ctgaggctga aatctgagcc 480
cctgggaatg agggaggagg agaaggcctg gatcctctgg tctgagggag gagaaaaagg 540
aaataacaga agtctgagac aaattttggt ctgtctagct ctgtaattaa ttaactaaag 600
tggatcaaat gagaaggtga aagttcacag aggaacactc ctgtctgtcg tcttggactg 660
ggtctccatc ccaccatcca gcgtcctggt ctacgaagag tccacaggga ccttgtgaag 720
aatcaacaag gcggggtcca gaggagtcac gtgtcccttc cactccgggt caccctgtcg 780
gaatgggggt cccgcggcct cagccctggg cgctggggct cctgctcttt ctccttcctg 840
ggagcctggg cgcaggtgag ggccgctccg ggccagggcc ctgctgcagg cgggcggcgg 900
gaggcggccc caggcaggca ggggcgaagc cagcgggacc cgagttcccc gcgagcccct 960
ggcgctgcct tctccgctca attaactttc tcaacgtctc ctgctgggcc tgaggctggg 1020
aaccacctgt cgcctcttgt gtctgtttcc ctctctctct ctgggtctct gtccccctct 1080
ctttctgggt ctcttgtctc tctctctctg ggtctctgtc cccccccccc cgggtttctg 1140
tcccctctct ctgaatctgt ccccctccct ccataataga ttcttctccc tccctgggta 1200
tctgtcccac tgcagtctag ttccccgccc gtgtgctccc ttcagctctg tttctgtctg 1260
cagaaagcca cctctccctc ctgtaccacc ttaccgcggt gtcctcgcct gccccgggga 1320
ctcctgcctt ctgggtgtcc ggctggctgg gcccgcagca gtacctgagc tacaatagcc 1380
tgcggggcga ggcggagccc tgtggagctt gggtctggga aaaccaggtg tcctggtatt 1440
gggagaaaga gaccacagat ctgaggatca aggagaagct ctttctggaa gctttcaaag 1500
ctttgggggg aaaaggtgag attccggtct ggaggggcaa ggggccgggt ccatgctccg 1560
gggccccgct tacctgtgtt tgggcgcccc aggtccctac actctgcagg gcctgctggg 1620
ctgtgaactg ggccctgaca acacctcggt gcccaccgcc aagttcgccc tgaacggcga 1680
ggagttcatg aatttcgacc tcaagcaggg cacctggggt ggggactggc ccgaggccct 1740
ggctatcagt cagcggtggc agcagcagga caaggcggcc aacaaggagc tcaccttcct 1800
gctattctcc tgcccgcacc gcctgcggga gcacctggag aggggccgcg gaaacctgga 1860
gtggaaggag cccccctcca tgcgcctgaa ggcccgaccc agcagccctg gcttttccgt 1920
gcttacctgc agcgccttct ccttctaccc tccggagctg caacttcggt tcctgcggaa 1980
tgggctggcc gctggcaccg gccagggtga cttcggcccc aacagtgacg gatccttcca 2040
cgcctcgtcg tcactaacag tcaaaagtgg cgatgagcac cactactgct gcattgtgca 2100
gcacgcgggg ctggcgcagc ccctcagggt ggagctgggt gaggtcccgc caggtggtga 2160
tgctcctggt ttcccgttgc cttgtctcac tgctgcgccg gtccttcctg agtctgacct 2220
tcctccccac tgctgccacc tccttgaatc tgactgcctt gaacctcacg cctgtcagtg 2280
cccccaaaac ctgatggctt gtccttccca aggccaactg ccttccgtct cctgctgctt 2340
ctggcctcac tgagtctgaa gagctgttaa ctaccatggc cagtcctccc tgagtctgac 2400
catcttccat cctgctgctg ctgctgctgc tgctgcgggt cttcctggaa tctgaccatt 2460
cgttgtctgc tatgcccgtc ctcaccaaga ctgactgcct gctgctttgc tactgcccgg 2520
gcccatgaga ctgacttccc actgctctgc ctgcctctcc ccactgcact ggcacagccc 2580
cgccttgccg ctgctgatcc attgccggtg tgaccgcggc cgctcgtgct gtagctggtt 2640
cttacgtcca acctgggggc agtctgccca gatcgcctgc ctggcctcgc ctctgcccat 2700
cagacacttg gtgctggaat ctccgaggct gggagggact gggagcgccc cagttctgtg 2760
tcctgactgg gtggggtgga ggggctgctc cacatctcac agcgttctct ggctgcagaa 2820
tctccagcca agtcctccgt gcctgtggtt ggaatcgttc ttggtttatt gctggtggta 2880
gtggccatcg caggcggtgt gctgttgtgg ggcaggatgc gcagcggtct gccaggtata 2940
tgcaggatgg gggaggggcg gggccgtgtg caggattggg tgggagtggc cttgtgcagg 3000
atgagggagg ggcggggccg tgtgcaggat tggggtgggg cgtggccgtg tgcagaatga 3060
gggaggggcg tggccgtgtg caggattggg gaggggaggg aaggggccgt gtgcaggatt 3120
ggggaggggc gtagctgtgt gcaggattgg ggaagggagg ggccgtgtgc aggattgggg 3180
gtcgtggcag gaagtagcga ctcagaaaga aagaggggag gcagagagat aatagagaca 3240
aaagagtccc tgaccaccct gggaaaagag atgggaacag agaccatagc gtgaccctgt 3300
caggtgtgtt gggtgggtga gaccttagat acttgtgctg acctctgttc ctctctctca 3360
gccccatggc tttctctcag cggcgatgac tctggtgacc tgttgcctgg tgggaacttg 3420
cccccagaag ctgaacctca aggtgcaaat gcctttccag ccacttcctg atgcagactc 3480
gggccccctg cccactgcag cctttcgggc tgtgtgacct cctgaactgt ctccgagcct 3540
cctgagggag cctgggcccg atgtcctccc atggatccct gcttttgtgg cctgcttcag 3600
tttcccttct taatgtacat ggttgttttc catctccaca taaatttggc cccaaatctg 3660
tgtgtgcatc gttattctca agtttcaagc agctggaata aattgaacgc gtctgggaaa 3720
gatcata 3727
<210> 2
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
agtggcatcc ccattccgac 20
<210> 3
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
agccagatct tctgtgcctg 20
<210> 4
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cttaaagtca cattcagcgt gcag 24
<210> 5
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tcaaggcagt cagattcaag gag 23
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
agcaccacta ctgctgcatt gt 22
<210> 7
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cttgagaata acgatgcaca cacag 25
<210> 8
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gaagtgggag gattaggtct gaac 24
<210> 9
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
aggctgcttc atccacagat tag 23
<210> 10
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cttaaagtca cattcagcgt gcag 24
<210> 11
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gagaaggtga aagttcacag aggaac 26
<210> 12
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tcaaggcagt cagattcaag gag 23
<210> 13
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
cttctccttg atcctcagat 20
<210> 14
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
agcaccacta ctgctgcatt gt 22
<210> 15
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atcagacact tggtgctgga atc 23
<210> 16
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
cttgagaata acgatgcaca cacag 25
Claims (4)
1.一种用于CRISPR/Cas9技术制备FcRn基因人源化小鼠模型的方法,其特征在于,包括以下步骤:
(1)构建表达针对鼠源FcRn基因的sgRNA的质粒;
(2)将人源FcRn基因的DNA序列、鼠源FcRn基因的3’同源臂和5’同源臂连接至质粒上,构建人源化FcRn基因的载体;
(3)将步骤(1)所述质粒体外转录获得的sgRNA与步骤(2)的载体以及Cas9 mRNA或Cas9蛋白注射至鼠受精卵细胞质或细胞核中,移植入受体动物母鼠生产FcRn基因人源化动物模型;
所述载体为包含DNA序列的载体,所述DNA序列中包含来自人源FcRn基因的部分Exon2、Intron2、Exon3、Intron3、Exon4、Exon5、Intron5、部分Exon6,以及来自鼠源FcRn基因的exon1、intron1、部分exon2、部分exon6、intron6、exon7,所述DNA序列如SEQ ID NO. 1所示;
其中针对鼠源SgRNA的序列如SEQ ID NO. 2和SEQ ID NO. 3所示。
2.如权利要求1所述的方法,其特征在于,进一步包括利用PCR扩增引物鉴定所述小鼠模型的步骤。
3.如权利要求1或2所述的方法,其特征在于,进一步包括利用测序引物鉴定所述小鼠模型的步骤。
4.权利要求1-3任一项所述的方法在制备人源化FcRn基因小鼠模型中的应用,所述应用为非疾病诊断或非疾病治疗目的。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110517875.9A CN113308476B (zh) | 2021-05-12 | 2021-05-12 | 一种FcRn基因人源化动物模型的构建方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110517875.9A CN113308476B (zh) | 2021-05-12 | 2021-05-12 | 一种FcRn基因人源化动物模型的构建方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113308476A CN113308476A (zh) | 2021-08-27 |
CN113308476B true CN113308476B (zh) | 2022-06-28 |
Family
ID=77373114
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110517875.9A Active CN113308476B (zh) | 2021-05-12 | 2021-05-12 | 一种FcRn基因人源化动物模型的构建方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113308476B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116114657B (zh) * | 2022-10-11 | 2024-06-11 | 上海交通大学医学院附属瑞金医院 | 一种红斑狼疮样小鼠模型的构建方法 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109022443A (zh) * | 2018-09-11 | 2018-12-18 | 江苏集萃药康生物科技有限公司 | 一种ctla4基因人源化动物模型的构建方法及其应用 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6744855B2 (ja) * | 2014-03-21 | 2020-08-19 | エフ.ホフマン−ラ ロシュ アーゲーF. Hoffmann−La Roche Aktiengesellschaft | 抗体のin vivoでの半減期のin vitroでの予測 |
-
2021
- 2021-05-12 CN CN202110517875.9A patent/CN113308476B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109022443A (zh) * | 2018-09-11 | 2018-12-18 | 江苏集萃药康生物科技有限公司 | 一种ctla4基因人源化动物模型的构建方法及其应用 |
Non-Patent Citations (3)
Title |
---|
"A single-valent long-acting human CD47 antagonist enhances antibody directed phagocytic activities";Fenglan Wu,et al.;《Cancer Immunol Immunother》;20200624;材料与方法部分 * |
"Homo sapiens Fc fragment of IgG, receptor, transporter, alpha, mRNA (cDNA clone IMAGE:2900361),BC020421.1";Strausberg,R.L.,et al.;《NCBI Genebank》;20031119;第1-2页 * |
"Humanized FcRn mouse models for evaluating pharmacokinetics of human IgG antibodies";Gabriele Proetzel and Derry C. Roopenian;《Methods》;20130716;第65卷(第1期);摘要,第2.1-2.2节,第3节 * |
Also Published As
Publication number | Publication date |
---|---|
CN113308476A (zh) | 2021-08-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6574811B2 (ja) | pH感受性免疫グロブリン配列を発現する非ヒト動物 | |
KR102186822B1 (ko) | 인간화 경쇄 마우스 | |
EP2954779B1 (en) | Mice that make heavy chain antibodies | |
EP3289869B1 (en) | Transgenic non-human animal expressing human-specific molecules and human fc gamma receptor family | |
JP2019068850A (ja) | ヒスチジン操作軽鎖抗体およびそれを生成するための遺伝子改変非ヒト動物 | |
CN103409448B (zh) | 一种模型小鼠制备方法及条件性细胞剔除重组载体 | |
CN109266656A (zh) | 一种PD1人源化BALB/c小鼠模型的构建方法及其应用 | |
CN108531487A (zh) | 人源化sirpa基因改造动物模型的制备方法及应用 | |
CN109486860B (zh) | 一种tigit人源化小鼠模型的构建方法及其应用 | |
CN117099741A (zh) | 具有人源化免疫球蛋白基因座的经遗传修饰的非人动物 | |
CA3144958A1 (en) | Transgenic mammals and methods of use thereof | |
CN113308476B (zh) | 一种FcRn基因人源化动物模型的构建方法 | |
CN112512310A (zh) | 用于无糖基化抗体产生的转基因小鼠和由此产生的无糖基化抗体的用途 | |
CN112626122B (zh) | hKDR人源化小鼠模型及其建立方法和应用 | |
CN109280674B (zh) | 一种筛选抗体的非人模式动物的构建方法及其应用 | |
CN111019972A (zh) | 一种cd27人源化小鼠模型的构建方法及其应用 | |
KR20200001022A (ko) | Irx1 유전자-녹아웃 형질전환 제브라피쉬 모델 및 이의 용도 | |
CN112608942B (zh) | Tnfrsf1b基因人源化动物模型的构建方法及应用 | |
CN114747541B (zh) | 一种psgl-1人源化非人类动物模型的构建方法及应用 | |
CN114786479B (zh) | 一种il-15人源化小鼠模型及其用途 | |
CN111705085B (zh) | 构建动物模型的方法及应用 | |
KR100443426B1 (ko) | Srg3 단백질을 t 세포 특이적으로 발현하는 형질전환동물 및 그의 제조방법 | |
CN115197942A (zh) | 一种可表达人源IgA1蛋白的鼠模型及其构建方法和应用 | |
WO2024115651A1 (en) | Rodents expressing a common light chain | |
AU2017261477A1 (en) | Mice that make heavy chain antibodies |
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 |