CN105210981A - 建立可应用于人类疾病研究的雪貂模型的方法及其应用 - Google Patents
建立可应用于人类疾病研究的雪貂模型的方法及其应用 Download PDFInfo
- Publication number
- CN105210981A CN105210981A CN201510587647.3A CN201510587647A CN105210981A CN 105210981 A CN105210981 A CN 105210981A CN 201510587647 A CN201510587647 A CN 201510587647A CN 105210981 A CN105210981 A CN 105210981A
- Authority
- CN
- China
- Prior art keywords
- ferret
- injection
- hcg
- fsh
- hour
- 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.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 56
- 238000011160 research Methods 0.000 title claims abstract description 14
- 238000011832 ferret model Methods 0.000 title claims abstract description 9
- 241000282341 Mustela putorius furo Species 0.000 claims abstract description 57
- 238000000338 in vitro Methods 0.000 claims abstract description 20
- 230000004720 fertilization Effects 0.000 claims abstract description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 13
- 201000010099 disease Diseases 0.000 claims abstract description 11
- 230000016087 ovulation Effects 0.000 claims abstract description 11
- 238000002347 injection Methods 0.000 claims description 26
- 239000007924 injection Substances 0.000 claims description 26
- 108090000623 proteins and genes Proteins 0.000 claims description 26
- 241001508687 Mustela erminea Species 0.000 claims description 19
- 108010062540 Chorionic Gonadotropin Proteins 0.000 claims description 18
- 102000011022 Chorionic Gonadotropin Human genes 0.000 claims description 18
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 18
- 229940084986 human chorionic gonadotropin Drugs 0.000 claims description 15
- 210000000130 stem cell Anatomy 0.000 claims description 12
- 108010000912 Egg Proteins Proteins 0.000 claims description 11
- 102000002322 Egg Proteins Human genes 0.000 claims description 11
- 210000004681 ovum Anatomy 0.000 claims description 11
- 210000003101 oviduct Anatomy 0.000 claims description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 8
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 claims description 8
- 239000012531 culture fluid Substances 0.000 claims description 7
- 101150088306 Disc1 gene Proteins 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 238000000520 microinjection Methods 0.000 claims description 6
- 238000013518 transcription Methods 0.000 claims description 6
- 230000035897 transcription Effects 0.000 claims description 6
- 238000002054 transplantation Methods 0.000 claims description 6
- 238000011067 equilibration Methods 0.000 claims description 5
- 230000009261 transgenic effect Effects 0.000 claims description 5
- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 4
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 claims description 4
- 229940052299 calcium chloride dihydrate Drugs 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 4
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- 230000035935 pregnancy Effects 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 239000001540 sodium lactate Substances 0.000 claims description 4
- 235000011088 sodium lactate Nutrition 0.000 claims description 4
- 229940005581 sodium lactate Drugs 0.000 claims description 4
- 229940054269 sodium pyruvate Drugs 0.000 claims description 4
- 108010003272 Hyaluronate lyase Proteins 0.000 claims description 3
- 102000001974 Hyaluronidases Human genes 0.000 claims description 3
- 210000001771 cumulus cell Anatomy 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 229960005210 follitropin alfa Drugs 0.000 claims description 3
- 108010006578 follitropin alfa Proteins 0.000 claims description 3
- 229960002773 hyaluronidase Drugs 0.000 claims description 3
- 238000010255 intramuscular injection Methods 0.000 claims description 3
- 239000007927 intramuscular injection Substances 0.000 claims description 3
- 210000000287 oocyte Anatomy 0.000 claims description 3
- 210000000582 semen Anatomy 0.000 claims description 3
- 210000000538 tail Anatomy 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 238000007877 drug screening Methods 0.000 claims description 2
- 210000004392 genitalia Anatomy 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 230000008961 swelling Effects 0.000 claims description 2
- 108091033409 CRISPR Proteins 0.000 abstract description 24
- 238000005516 engineering process Methods 0.000 abstract description 16
- 238000010354 CRISPR gene editing Methods 0.000 abstract description 10
- 230000006698 induction Effects 0.000 abstract description 6
- 241001465754 Metazoa Species 0.000 description 14
- 210000004556 brain Anatomy 0.000 description 12
- 101150061941 Dcx gene Proteins 0.000 description 10
- 206010064571 Gene mutation Diseases 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 208000012902 Nervous system disease Diseases 0.000 description 5
- 238000010171 animal model Methods 0.000 description 4
- 210000001161 mammalian embryo Anatomy 0.000 description 4
- 210000001672 ovary Anatomy 0.000 description 4
- 210000001082 somatic cell Anatomy 0.000 description 4
- 241000282339 Mustela Species 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 230000004641 brain development Effects 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000003710 cerebral cortex Anatomy 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000008034 disappearance Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000012173 estrus Effects 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 208000004141 microcephaly Diseases 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000010449 nuclear transplantation Methods 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 210000002394 ovarian follicle Anatomy 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- -1 Aspm Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 241000543821 Oestrus Species 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 206010042573 Superovulation Diseases 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 101150010487 are gene Proteins 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000002490 cerebral effect Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 230000001149 cognitive effect Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000008451 emotion Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000003195 fascia Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000009027 insemination Effects 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000015654 memory Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000008271 nervous system development Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 201000000980 schizophrenia Diseases 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000008010 sperm capacitation Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
Classifications
-
- 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
- A01K67/02—Breeding vertebrates
-
- 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
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Environmental Sciences (AREA)
- Biodiversity & Conservation Biology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Animal Husbandry (AREA)
- Animal Behavior & Ethology (AREA)
- Developmental Biology & Embryology (AREA)
- Gynecology & Obstetrics (AREA)
- Reproductive Health (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种建立可应用于疾病研究的雪貂模型的方法及其应用，属于生物学领域，具体涉及一种雪貂促排卵技术和一种雪貂体外受精技术，以及基于上述方面的利用CRISPR/Cas9的技术建立雪貂模型的方法，所述方法可应用于人类疾病研究。
Description
技术领域
本发明属于生物学领域，具体涉及一种雪貂促排卵技术和一种雪貂体外受精技术，以及基于上述方面的利用CRISPR/Cas9的技术建立雪貂模型的方法，所述方法可应用于系统研究人类疾病。
背景技术
大脑是人类控制认知、记忆、情感和活动的功能器官，某些与大脑发育相关基因的缺失或突变直接导致神经疾病的发生。高等哺乳动物的大脑皮层具有沟和回的结构，这一结构大大的增加了大脑皮层的表面积，研究表明这与高级认知等大脑功能密切相关。
雪貂作为新型实验动物，已经被广泛的应用于呼吸道疾病等的研究，但是并没有在神经系统发育中普遍应用。虽然雪貂不属于灵长类动物，但是它与平滑脑的小鼠相比，雪貂的大脑具有沟和回的结构，并且是有社会性的动物，因此，应用雪貂作为模式动物，研究直接大脑发育型疾病以及精神类疾病的发病机理和临床治疗等，具有很大的现实意义。此外，雪貂还具有体形小、易饲养、繁殖周期短、一胎多崽的优点，是研究神经系统疾病发病机理的首选模式动物。
雪貂之所以没有被神经科学研究广泛应用，其中主要原因是转基因动物无法实现。随着CRISPR/Cas9技术的发展，转基因动物制备变得相对简便[1,2]。
CRISPR/Cas9这一编辑基因的技术已经广泛应用于各种物种，包括小鼠、大鼠、猴等。以小鼠为例，主要实验过程如图1所示，将一个或者多个sgRNA(单向导RNA)和Cas9mRNA注射到受精卵里，sgRNA介导Cas9核酸酶在小鼠受精卵的特定基因组位点上进行切割、修复，导致基因被改变。
由于雪貂作为实验动物并没有被广泛应用，鲜有关于转基因雪貂的报道。雪貂作为实验动物的驯化和饲养条件要求比较严苛，而且生殖周期等生理习性并没有非常详尽的研究资料，目前已经报道的唯一一例转基因雪貂，是通过病毒结合核移植的方法获得的[3,4]，主要方法过程如图2所示。正常的雪貂体细胞经过定向基因修饰变为改变遗传学特性的修饰后体细胞，然后在成熟的雪貂卵母细胞中通过显微操作的方法用修饰后的体细胞的细胞核替换卵母细胞的细胞核。核移植后的卵母细胞经过发育，胚胎的基因就可以得到相应的改变。
然而，一方面，CRISPR/Cas9作为新兴的技术，从未应用于雪貂这种模式动物；另一方面，病毒结合体细胞核移植的方法比较复杂，操作性不高，效率低下，只有1-3％，而且使转基因动物有可能带上病毒的风险，此外，目前已经报道的雪貂的超排方案是针对MarshallFerret的，用于AngoraFerret超排效率非常低，卵子不成熟，且无法做到雪貂的体外受精，无法满足高效制备转基因雪貂的要求。
发明内容
本发明针对现有技术中建立可应用于人类疾病研究的雪貂模型的方法的不足，一方面提供一种雪貂促排卵技术和一种雪貂体外受精技术，另一方面，在上述基础上，利用CRISPR/Cas9的技术，以3种基因(这三个基因都与神经系统疾病相关，一个是平滑脑症、一个是头小畸形症、一个是精神分裂症)为例，建立可应用于神经系统疾病研究的雪貂模型的方法，并将其用于相关疾病的致病机理研究，相关药物的筛选和安全性评价，以及临床手术治疗研究的模式动物。
本发明首次应用PMSG，FSH和HCG联合超排卵方法结合体外受精技术，使得转基因雪貂的制备工作可以在尽量少的耗损实验动物的基础上顺利开展。在取得的雪貂受精卵中，本发明首次应用CRISPR/Cas9技术，对受精卵的特定的与神经系统疾病直接相关的基因，包括Dcx,Aspm,Disc1进行编辑，从而最终导致这三个基因突变，无法正常行使该基因的生物学功能，获得类似于人类由于这三个基因突变而导致的神经系统疾病。
具体而言，本发明包括以下方面：
1.一种促进雪貂排卵的方法，所述方法使用PMSG(PregnantMareSerumGonadotropin)、FSH(FollitropinAlfa)和HCG(HumanChorionicGonadotropin)联合进行。
2.根据第1项所述的方法，所述方法包括以下步骤：
1)给母貂腹腔注射PMSG，优选注射200-300单位，更优选300单位；
2)24-48小时后，肌肉注射第一针FSH，此后连续注射8-10天FSH，每次的FSH注射量优选为5-10单位，更优选为10单位；
3)如果外阴肿胀，颜色由红变白则腹腔注射HCG，HCG的注射量优选为200-300单位，更优选为300单位；
4)取卵，取卵时间优选在HCG注射后40-48小时，更优选为HCG注射后48小时。
3.根据第2项所述的方法，第1)步和第2)步之间间隔48小时。
4.根据第2项所述的方法，FSH的注射为每天两次，间隔12小时。
5.一种雪貂体外授精方法，所述方法包括：
1)取卵：手术取得雪貂的输卵管，用预热的HCZB培养液经输卵管伞口冲出卵丘卵母细胞复合体，用透明质酸酶消化成无卵丘细胞的单个卵母细胞，放入38.5℃，5％CO2预平衡3小时的IVF培养滴中备用；
2)体外受精：将公貂经附睾尾取出精液马上放入步骤1)中获得的含有卵母细胞的IVF培养滴中，共孵育，完成体外受精。
6.根据第5项所述的方法，所述共孵育时间为3-4小时。
7.根据第5项所述的方法，所述HCZB培养液的组成为：81.62mM氯化钠,4.83mM氯化钾,1.18mM磷酸二氢钾,1.18mM硫酸镁,5mM碳酸氢钠,1.7mM二水合氯化钙,31.3mM乳酸钠,0.27mM丙酮酸钠,20mMHepes,1mM谷氨酰胺,0.1mMEDTA2Na,5.5mM葡萄糖,0.007％PVA,1N盐酸。
8.一种建立雪貂模型的方法，所述方法包括以下步骤：
1)针对目的基因设计sgRNA单向导RNA序列，并体外转录CAS9mRNA和sgRNA序列；
2)根据1-4任一项所述的方法促雪貂排卵；
3)根据5-7任一项所述的方法进行雪貂体外受精；
4)将步骤1)获得的Cas9mRNA和目的基因的sgRNA显微注射入受精卵；
5)将步骤4)获得的受精卵移植入受体进行妊娠；
6)鉴定子代转基因雪貂。
9.根据第8项所述的方法，所述目的基因选自Aspm,Dcx,Disc1。
10.根据第8或9项所述的方法用于相关人类疾病研究及针对人类疾病的药物筛选和/或安全性评价方面的用途。
本发明首次使用PMSG，FSH和HCG联合促排卵，排卵稳定高效，达到25-35枚/只。而现有技术中仅有的可参考的超排方案是使用PMSG和HCG联合超排方案，效率低，卵不成熟。此外，本发明首次使用雪貂的体外受精而不需要体外获能。另外本发明首次应用CRISPR/Cas9技术制备的转基因雪貂，相比之前唯一一例应用病毒结合体细胞核移植的方法，效率提高很多，可以达到80％左右，并且没有病毒危害性。本发明的模型可以例如模拟人由于Dcx基因突变或者Aspm基因突变引起的神经疾病，成为最为合适的疾病模式动物。而之前已有的Dcx突变小鼠模型，因小鼠的大脑没有沟回，故不能复制人的疾病表型。
附图说明
图1.应用CRISPR/Cas9技术制备转基因小鼠示意图。
图2.通过基因编辑体细胞核结合核移植的方法改变胚胎基因组的示意图。图3.sgRNA的设计，其中对于每个基因设计了2个sgRNA,其中灰色带下划线的GGT/GGA/GGG/GGC序列为Protospacer-adjacentmotif(PAM)，其余灰色不带下划线的为基因干扰靶序列。
图4.三个不同品系的转基因雪貂经过T7EN1酶切鉴定，可以被酶切的是基因突变雪貂，被星号标出。
图5.三个不同品系的转基因雪貂经过测序分析具体的基因突变位点。与野生型比较，删除的碱基用点表示，增加的碱基用小写字母标注。同时，括号内表示缺失了或者增加了几个碱基，以及这种类型的结果在20个检测中所占数量。
图6.Dcx转基因雪貂的大脑结构改变。转基因雪貂大脑皮层变薄、沟回变少、脑室变大。
图7.Aspm转基因雪貂的大脑结构改变。转基因雪貂大脑变小、沟回变浅。
具体实施方式
实施例1.CRISPR/Cas9靶向修饰基因载体构建和体外转录
1)sgRNA转录载体的构建：针对雪貂Aspm(GenBankAccessionNo:XM_004756200),Dcx(GenBankAccessionNo:XM_004769082),Disc1(GenBankAccessionNo:XM_013047589)三个基因，设计了特异的sgRNA序列(图3)，具体序列参见表1。
表1
每2条单链核酸序列(表2)退火形成双链DNA，双链DNA连接到px330(Addgene,42230)载体中。
表2.sgRNA克隆引物序列
2)Cas9和sgRNA体外转录：利用表3中的引物将T7转录子通过PCR方法加入到Cas9和sgRNA的转录起始位点，Cas9序列和参考文献[2]中一致，PCR产物经过回收净化，用mMESSAGEmMACHINET7ULTRA试剂盒(LifeTechnologies)进行体外转录。转录产生的Cas9mRNA和sgRNA用MEGAclear试剂盒(LifeTechnologies)纯化并测量浓度。
表3.连接T7转录子引物
实施例2.雪貂促排卵
选择2-3岁，体重1.5-2KG，3周左右未发情经产母貂，腹腔注射300单位PMSG(PregnantMareSerumGonadotropin)(宁波三生药业)，48小时后肌肉注射第一针FSH(FollitropinAlfa)(MerckSerono)10单位，此后连续注射10天，每天两次，间隔12小时，每次10单位，在注射FSH后持续观察雪貂发情情况，如果发情，则腹腔注射300单位HCG(HumanChorionicGonadotropin)(MerckSerono)。注射HCG48小时后取卵。本方法排卵稳定高效，达到25-35枚/只，在AngoraFerret中应用，比之前文献报道的促排卵方法(无成熟的卵母细胞)提高效率约100％，并且均为成熟的卵母细胞。
我们还使用其它PMSG、FSH和HCG浓度(如200、250单位的PMSG，5、8单位的FSH，200、250单位的HCG)，以及FSH注射前的其它间隔时间(如24、36小时)、HCG注射和取卵间的其它间隔时间(如40、44小时)，均获得了与上述相似的促排卵效果。
实施例3.雪貂体外授精
1)取卵：手术取得雪貂的输卵管，用预热的HCZB培养液(81.62mM氯化钠,4.83mM氯化钾,1.18mM磷酸二氢钾,1.18mM硫酸镁,5mM碳酸氢钠,1.7mM二水合氯化钙,31.3mM乳酸钠,0.27mM丙酮酸钠,20mMHepes,1mM谷氨酰胺,0.1mMEDTA2Na,5.5mM葡萄糖,0.007％PVA,1N盐酸)经输卵管伞口冲出卵丘卵母细胞复合体，用透明质酸酶消化成无卵丘细胞的单个卵母细胞，放入38.5℃，5％CO2预平衡3小时的IVF培养滴(LifeTechnologies)中备用。
2)体外受精：选择3-4岁，体重2-4KG的健康公貂，经附睾尾取出精液马上放入含有卵母细胞的IVF培养滴中，共孵育3-4小时，完成体外受精。
实施例4.雪貂受精卵显微注射
1)体外受精的受精卵：在授精3-4小时后，将卵母细胞自IVF培养基中取出，移入显微操作的液滴中，用Piezo(Narishige)将混合好的Cas9mRNA(100ng/μl)和目的基因的sgRNAs(50ng/μl)显微注射入卵细胞质内，每枚注射量相当于一个原核的体积。注射结束后室温恢复15分钟，移入38.5°C，5％CO2预平衡好的CZB培养液(81.62mM氯化钠,4.83mM氯化钾,1.18mM磷酸二氢钾,1.18mM硫酸镁,25mM碳酸氢钠,0.1mMEDTA2Na,5.5mM葡萄糖,31.3mM乳酸钠,1.7mM二水合氯化钙,0.27mM丙酮酸钠,1mM谷氨酰胺,5g/l牛血清白蛋白)液滴中备用。
2)正常受精的受精卵：取正常合笼40-48小时后的母貂，手术取得输卵管，经输卵管伞口冲出受精卵，移入显微操作液滴中，用Piezo将混合好的Cas9mRNA(100ng/μl)和sgRNAs(50ng/μl)显微注射入卵细胞质内，每枚注射量相当于一个原核的体积。注射结束后室温恢复15分钟，移入38.5℃，5％CO2预平衡好的CZB培养液液滴中备用。
实施例5.受精卵移植入受体
将自然发情且与结扎公貂合笼24小时的母貂麻醉，使其侧卧于手术台上，在其左侧卵巢上方开2-3厘米的口，把卵巢拉到体外，经输卵管伞口移入显微注射过的胚胎15-18枚。并且观察卵巢是否已经排卵，若有明显未排的卵泡，则用1ML注射器将卵泡挑破。然后将卵巢恢复原位，手术缝合伤口。并注射20-30单位LH(LuteinizingHormone)(宁波三生药业)辅助着床。在胚胎移植后21天可用手检查母貂是否已经妊娠。
实施例6.转基因雪貂的鉴定
手术后母貂单独饲养，在胚胎移植后21天可用手检查母貂是否已经妊娠。母貂自然分娩，出生2周后，小貂进行编号并取尾部组织并对小貂的基因型进行鉴定。组织经速冻研磨裂解，提取基因组后，对于干扰的目的基因进行PCR扩增。PCR扩增引物见表4，PCR扩增产物经过T7EN1限制性内切酶(NEB)处理后，经过凝胶电泳分离，如果小貂基因被改变，可以在凝胶电泳图中看到一系列低分子量的条带。应用本方法，可以鉴定出目的基因被改变的小貂，用星号标示(图4)。此外，将确定为转基因雪貂的目的基因用上述方法和引物(表4)，进行PCR片段扩增，PCR产物克隆到pMD-19T载体(Takara)中并进行测序，获得被改变后基因组的序列(图5)。与野生型比较，Dcx基因在不同的小貂中被删除4－98个碱基，或者增加1－2个碱基。即使在同一只小貂里，Dcx基因也有不同的编辑方式。Aspm基因在不同的小貂里缺失了2－15个碱基。我们仅获得了一只Disc1转基因小貂，Disc1基因增加了1个碱基或者缺失了3个碱基。
表4.转基因雪貂基因型鉴定PCR引物
实施例7.转基因雪貂的相关表型分析
由于Dcx基因与人类大脑沟回的产生密切相关，因此，应用小动物磁共振成像设备(Siemens)，对于14周大的幼年雪貂进行脑结构的成像分析。发现在Dcx基因被突变后的幼年雪貂的大脑呈现了沟回明显缺失和脑腔变大的表型(图6)。此外，Aspm基因与人脑小畸形症直接相关，经过小动物磁共振成像技术检测，幼年雪貂在Aspm基因突变后，也呈现了脑体积减小和沟回变浅的表型(图7)。
参考文献
1.Cell.2013Sep12；154(6):1370-9.One-stepgenerationofmicecarryingreporterandconditionalallelesbyCRISPR/Cas-mediatedgenomeengineering.YangH,WangH,ShivalilaCS,ChengAW,ShiL,JaenischR.
2.Cell.2013May9；153(4):910-8.One-stepgenerationofmicecarryingmutationsinmultiplegenesbyCRISPR/Cas-mediatedgenomeengineering.WangH,YangH,ShivalilaCS,DawlatyMM,ChengAW,ZhangF,JaenischR.3.JClinInvest.2008Apr；118(4):1578-83.Adeno-associatedvirus-targeteddisruptionoftheCFTRgeneinclonedferrets.SunX,YanZ,YiY,LiZ,LeiD,RogersCS,ChenJ,ZhangY,WelshMJ,LenoGH,EngelhardtJF.
4.DevBiol.2006May15；293(2):439-48.Clonedferretsproducedbysomaticcellnucleartransfer.LiZ,SunX,ChenJ,LiuX,WiselySM,ZhouQ,RenardJP,LenoGH,EngelhardtJF.
5.ReprodBiolEndocrinol.2003Nov7；1:83.Progresstowardgeneratingaferretmodelofcysticfibrosisbysomaticcellnucleartransfer.LiZ,EngelhardtJF。
Claims (10)
1.一种促进雪貂排卵的方法，所述方法使用PMSG(PregnantMareSerumGonadotropin)、FSH(FollitropinAlfa)和HCG(HumanChorionicGonadotropin)联合进行。
2.根据权利要求1所述的方法，所述方法包括以下步骤：
1)给母貂腹腔注射PMSG，优选注射200-300单位，更优选300单位；
2)24-48小时后，肌肉注射第一针FSH，此后连续注射8-10天FSH，每次的FSH注射量优选为5-10单位，更优选为10单位；
3)如果外阴肿胀，颜色由红变白则腹腔注射HCG，HCG的注射量优选为200-300单位，更优选为300单位；
4)取卵，取卵时间优选在HCG注射后40-48小时，更优选为HCG注射后48小时。
3.根据权利要求2所述的方法，第1)步和第2)步之间间隔24－48小时。
4.根据权利要求2所述的方法，FSH的注射为每天两次，间隔12小时。
5.一种雪貂体外授精方法，所述方法包括：
1)取卵：手术取得雪貂的输卵管，用预热的HCZB培养液经输卵管伞口冲出卵丘卵母细胞复合体，用透明质酸酶消化成无卵丘细胞的单个卵母细胞，放入38.5℃，5％CO2预平衡3小时的IVF培养滴中备用；
2)体外受精：将公貂，经附睾尾取出精液马上放入步骤1)中获得的含有卵母细胞的IVF培养滴中，共孵育，完成体外受精。
6.根据权利要求5所述的方法，所述共孵育时间为3-4小时。
7.根据权利要求5所述的方法，所述HCZB培养液的组成为：81.62mM氯化钠,4.83mM氯化钾,1.18mM磷酸二氢钾,1.18mM硫酸镁,5mM碳酸氢钠,1.7mM二水合氯化钙,31.3mM乳酸钠,0.27mM丙酮酸钠,20mMHepes,1mM谷氨酰胺,0.1mMEDTA2Na,5.5mM葡萄糖,0.007％PVA,1N盐酸。
8.一种建立雪貂模型的方法，所述方法包括以下步骤：
1)针对目的基因设计sgRNA单向导RNA序列，并体外转录CAS9mRNA和sgRNA序列；
2)根据权利要求1-4任一项所述的方法促雪貂排卵；
3)根据权利要求5-7任一项所述的方法进行雪貂体外受精；
4)将步骤1)获得的Cas9mRNA和目的基因的sgRNA显微注射入受精卵；
5)将步骤4)获得的受精卵移植入受体进行妊娠；
6)鉴定子代转基因雪貂。
9.根据权利要求8所述的方法，所述目的基因选自Aspm,Dcx,Disc1。
10.根据权利要求8或9所述的方法用于人类疾病研究及针对人类疾病的药物筛选和/或安全性评价方面的用途。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510587647.3A CN105210981B (zh) | 2015-09-15 | 2015-09-15 | 建立可应用于人类疾病研究的雪貂模型的方法及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510587647.3A CN105210981B (zh) | 2015-09-15 | 2015-09-15 | 建立可应用于人类疾病研究的雪貂模型的方法及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105210981A true CN105210981A (zh) | 2016-01-06 |
| CN105210981B CN105210981B (zh) | 2018-09-28 |
Family
ID=54980604
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510587647.3A Active CN105210981B (zh) | 2015-09-15 | 2015-09-15 | 建立可应用于人类疾病研究的雪貂模型的方法及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105210981B (zh) |
Cited By (26)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10227581B2 (en) | 2013-08-22 | 2019-03-12 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| CN109777828A (zh) * | 2018-12-28 | 2019-05-21 | 江苏集萃药康生物科技有限公司 | 一种基于IVF与CRISPR/Cas9基因编辑技术的基因改造动物模型制备方法 |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| CN111011295A (zh) * | 2019-11-25 | 2020-04-17 | 芜湖职业技术学院 | 一种提高北极狐繁殖的方法 |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| CN112715475A (zh) * | 2020-12-28 | 2021-04-30 | 无锡珊瑚礁生物科技有限公司 | 一种提高雪貂产仔率的方法 |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1436237A (zh) * | 2000-05-05 | 2003-08-13 | 农业研究有限公司 | 与提高或降低哺乳动物排卵率有关的核苷酸序列 |
| US20080299077A1 (en) * | 2007-06-01 | 2008-12-04 | Nevada Cancer Institute | Isolation and growth of stem cells from hemangiomas |
| US20120180146A1 (en) * | 2003-08-22 | 2012-07-12 | The Jackson Laboratory | Methods for maintaining genetic stability of inbred animal strains |
| CN103348012A (zh) * | 2010-11-27 | 2013-10-09 | 朱坚 | 一种人源化的转基因动物 |
| CN103478071A (zh) * | 2013-06-28 | 2014-01-01 | 四川农业大学 | 一种诱导糖尿病模型的建立方法 |
| CN103566353A (zh) * | 2013-11-06 | 2014-02-12 | 刘志刚 | 一种构建哮喘动物模型的方法 |
| CN103740639A (zh) * | 2013-09-02 | 2014-04-23 | 北京大学人民医院 | 构建人源化Ph染色体阳性急性淋巴细胞白血病小鼠模型的方法 |
| CN104560944A (zh) * | 2014-12-24 | 2015-04-29 | 张华� | 一种hla-dqbi完全基因剔除实验小鼠模型的建立方法 |
-
2015
- 2015-09-15 CN CN201510587647.3A patent/CN105210981B/zh active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1436237A (zh) * | 2000-05-05 | 2003-08-13 | 农业研究有限公司 | 与提高或降低哺乳动物排卵率有关的核苷酸序列 |
| US20120180146A1 (en) * | 2003-08-22 | 2012-07-12 | The Jackson Laboratory | Methods for maintaining genetic stability of inbred animal strains |
| US20080299077A1 (en) * | 2007-06-01 | 2008-12-04 | Nevada Cancer Institute | Isolation and growth of stem cells from hemangiomas |
| CN103348012A (zh) * | 2010-11-27 | 2013-10-09 | 朱坚 | 一种人源化的转基因动物 |
| CN103478071A (zh) * | 2013-06-28 | 2014-01-01 | 四川农业大学 | 一种诱导糖尿病模型的建立方法 |
| CN103740639A (zh) * | 2013-09-02 | 2014-04-23 | 北京大学人民医院 | 构建人源化Ph染色体阳性急性淋巴细胞白血病小鼠模型的方法 |
| CN103566353A (zh) * | 2013-11-06 | 2014-02-12 | 刘志刚 | 一种构建哮喘动物模型的方法 |
| CN104560944A (zh) * | 2014-12-24 | 2015-04-29 | 张华� | 一种hla-dqbi完全基因剔除实验小鼠模型的建立方法 |
Non-Patent Citations (2)
| Title |
|---|
| 林筱璐等: "不同因素对猪卵母细胞体外成熟培养的影响", 《中国畜牧兽医》 * |
| 王新蕾等: "动物超数排卵技术研究进展", 《实验动物与比较医学》 * |
Cited By (51)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US12006520B2 (en) | 2011-07-22 | 2024-06-11 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
| US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US10227581B2 (en) | 2013-08-22 | 2019-03-12 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US9737604B2 (en) | 2013-09-06 | 2017-08-22 | President And Fellows Of Harvard College | Use of cationic lipids to deliver CAS9 |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
| US10077453B2 (en) | 2014-07-30 | 2018-09-18 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US12043852B2 (en) | 2015-10-23 | 2024-07-23 | President And Fellows Of Harvard College | Evolved Cas9 proteins for gene editing |
| US10167457B2 (en) | 2015-10-23 | 2019-01-01 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US11999947B2 (en) | 2016-08-03 | 2024-06-04 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US12084663B2 (en) | 2016-08-24 | 2024-09-10 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| CN109777828A (zh) * | 2018-12-28 | 2019-05-21 | 江苏集萃药康生物科技有限公司 | 一种基于IVF与CRISPR/Cas9基因编辑技术的基因改造动物模型制备方法 |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| CN111011295A (zh) * | 2019-11-25 | 2020-04-17 | 芜湖职业技术学院 | 一种提高北极狐繁殖的方法 |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| US12031126B2 (en) | 2020-05-08 | 2024-07-09 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| CN112715475B (zh) * | 2020-12-28 | 2023-11-24 | 无锡珊瑚礁生物科技有限公司 | 一种提高雪貂产仔率的方法 |
| CN112715475A (zh) * | 2020-12-28 | 2021-04-30 | 无锡珊瑚礁生物科技有限公司 | 一种提高雪貂产仔率的方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105210981B (zh) | 2018-09-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105210981A (zh) | 建立可应用于人类疾病研究的雪貂模型的方法及其应用 | |
| CN108660161B (zh) | 基于CRISPR/Cas9技术的制备无嵌合基因敲除动物的方法 | |
| CN107043787B (zh) | 一种基于CRISPR/Cas9获得MARF1定点突变小鼠模型的构建方法和应用 | |
| CN105132427B (zh) | 一种以RNA介导的特异性敲除双基因获得基因编辑绵羊的方法及其专用sgRNA | |
| Takahashi et al. | Birth of healthy offspring following ICSI in in vitro-matured common marmoset (Callithrix jacchus) oocytes | |
| CN109706184B (zh) | 自闭症模型犬的建立方法 | |
| CN105505879B (zh) | 一种培养转基因动物胚胎细胞或转基因动物的方法及培养基 | |
| CN106119284A (zh) | 一种用于构建免疫缺陷动物模型的产品及其应用 | |
| CN106282231A (zh) | 粘多糖贮积症ii型动物模型的构建方法及应用 | |
| CN110029087A (zh) | 一种体外人睾丸精子发生模型的制备方法 | |
| CN113088521A (zh) | 一种基于CRISPR/Cas9技术的Ahnak2基因敲除动物模型的构建方法 | |
| CN101878747A (zh) | 一种RdRp可调控表达和活体观测的小鼠模型的构建方法 | |
| CN110129320A (zh) | 一种获得基因编辑绵羊的方法及其专用sgRNA和Oligo DNA | |
| CN111500580B (zh) | 一种基因编辑方法 | |
| CN108624621B (zh) | 非人灵长类的体细胞克隆动物的制备方法 | |
| CN105132426A (zh) | 一种以RNA介导的特异性敲除FGF5基因获得基因编辑绵羊的方法及其专用sgRNA | |
| CN114868707B (zh) | 一种代谢性脑病和心律失常疾病的斑马鱼模型及其应用 | |
| Sepulveda-Rincon et al. | Embryo cell allocation patterns are not altered by biopsy but can be linked with further development | |
| CN111518839B (zh) | 一种等位特异性位点编辑方法 | |
| Briski et al. | Comparison of ICSI, IVF, and in vivo derived embryos to produce CRISPR-Cas9 gene-edited pigs for xenotransplantation | |
| CN109777828A (zh) | 一种基于IVF与CRISPR/Cas9基因编辑技术的基因改造动物模型制备方法 | |
| CN112342249A (zh) | Uox基因敲除小鼠模型及其构建方法 | |
| CN114292880B (zh) | 斑马鱼vps28基因在制备神经血管单元血管障碍模型中的应用 | |
| CN103923215B (zh) | 使ACC-α基因启动子PⅢ失活的物质及其应用 | |
| Huang et al. | Interspecies blastocyst complementation generates functional rat cell-derived forebrain tissues in mice |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |