CN114410691B - Slc35e1基因敲除小鼠动物模型的构建方法和应用 - Google Patents
Slc35e1基因敲除小鼠动物模型的构建方法和应用 Download PDFInfo
- Publication number
- CN114410691B CN114410691B CN202210168465.2A CN202210168465A CN114410691B CN 114410691 B CN114410691 B CN 114410691B CN 202210168465 A CN202210168465 A CN 202210168465A CN 114410691 B CN114410691 B CN 114410691B
- Authority
- CN
- China
- Prior art keywords
- mouse
- slc35e1
- gene
- animal model
- gene knockout
- 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
- 241000699666 Mus <mouse, genus> Species 0.000 title claims abstract description 76
- 108091006952 SLC35E1 Proteins 0.000 title claims abstract description 60
- 102100032275 Solute carrier family 35 member E1 Human genes 0.000 title claims abstract description 41
- 238000003209 gene knockout Methods 0.000 title claims abstract description 29
- 238000010171 animal model Methods 0.000 title claims abstract description 26
- 238000010276 construction Methods 0.000 title claims abstract description 11
- 235000013601 eggs Nutrition 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
- 108091033409 CRISPR Proteins 0.000 claims abstract description 12
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 10
- 238000000338 in vitro Methods 0.000 claims abstract description 5
- 101710163270 Nuclease Proteins 0.000 claims abstract description 4
- 108020004414 DNA Proteins 0.000 claims description 14
- 238000012163 sequencing technique Methods 0.000 claims description 8
- 238000000520 microinjection Methods 0.000 claims description 6
- 238000010354 CRISPR gene editing Methods 0.000 claims description 4
- 238000005516 engineering process Methods 0.000 claims description 4
- 230000013011 mating Effects 0.000 claims description 3
- 210000003101 oviduct Anatomy 0.000 claims 1
- 201000004681 Psoriasis Diseases 0.000 abstract description 18
- 208000027531 mycobacterial infectious disease Diseases 0.000 abstract description 15
- 239000003814 drug Substances 0.000 abstract description 6
- 238000012216 screening Methods 0.000 abstract description 6
- 230000007246 mechanism Effects 0.000 abstract description 3
- 230000009471 action Effects 0.000 abstract description 2
- 238000011160 research Methods 0.000 description 9
- 206010024229 Leprosy Diseases 0.000 description 8
- 238000000034 method Methods 0.000 description 7
- 108020005004 Guide RNA Proteins 0.000 description 6
- 108091006185 SLC35 Proteins 0.000 description 6
- 102000037053 SLC35 Human genes 0.000 description 5
- 208000015181 infectious disease Diseases 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 241000251468 Actinopterygii Species 0.000 description 3
- 206010062207 Mycobacterial infection Diseases 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000006071 cream Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 229960002751 imiquimod Drugs 0.000 description 3
- DOUYETYNHWVLEO-UHFFFAOYSA-N imiquimod Chemical compound C1=CC=CC2=C3N(CC(C)C)C=NC3=C(N)N=C21 DOUYETYNHWVLEO-UHFFFAOYSA-N 0.000 description 3
- 238000011532 immunohistochemical staining Methods 0.000 description 3
- 230000008506 pathogenesis Effects 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical class [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 208000031998 Mycobacterium Infections Diseases 0.000 description 2
- 241000187492 Mycobacterium marinum Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108091006953 SLC35E2B Proteins 0.000 description 2
- 102100033783 Solute carrier family 35 member E2B Human genes 0.000 description 2
- 229940069428 antacid Drugs 0.000 description 2
- 239000003159 antacid agent Substances 0.000 description 2
- 230000001458 anti-acid effect Effects 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000011813 knockout mouse model Methods 0.000 description 2
- 230000003990 molecular pathway Effects 0.000 description 2
- 201000000585 muscular atrophy Diseases 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 208000013135 GNE myopathy Diseases 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000186359 Mycobacterium Species 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 102000003480 SLC35D Human genes 0.000 description 1
- 108060008450 SLC35D Proteins 0.000 description 1
- 108091006957 SLC35D1 Proteins 0.000 description 1
- 108091006960 SLC35D2 Proteins 0.000 description 1
- 102000003479 SLC35E Human genes 0.000 description 1
- 108060008451 SLC35E Proteins 0.000 description 1
- 101150107569 SLC35E2B gene Proteins 0.000 description 1
- 102100032285 UDP-N-acetylglucosamine/UDP-glucose/GDP-mannose transporter Human genes 0.000 description 1
- 102100032284 UDP-glucuronic acid/UDP-N-acetylgalactosamine transporter Human genes 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 101150038500 cas9 gene Proteins 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 208000018631 connective tissue disease Diseases 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 210000002288 golgi apparatus Anatomy 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 206010040882 skin lesion Diseases 0.000 description 1
- 231100000444 skin lesion Toxicity 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012795 verification Methods 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
- C12N15/87—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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- C—CHEMISTRY; METALLURGY
- 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
- A01K2267/0337—Animal models for infectious diseases
-
- 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
- A01K2267/035—Animal model for multifactorial diseases
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Environmental Sciences (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Veterinary Medicine (AREA)
- Endocrinology (AREA)
- Plant Pathology (AREA)
- Public Health (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Rheumatology (AREA)
- Pathology (AREA)
- Epidemiology (AREA)
- Diabetes (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了SLC35E1基因敲除小鼠动物模型的构建方法和应用,属于生物技术领域。构建方法包括:根据SLC35E1基因序列确定SLC35E1小鼠待敲除基因的两对特异性靶点gRNA1和gRNA2、gRNA3和gRNA4,并与Cas9核酸酶体外转录成mRNA,显微注射入小鼠受精卵并移植至代孕母鼠输卵管内,待产出小鼠后传代培养,经基因型鉴定得到稳定遗传的SLC35E1基因敲除小鼠动物模型。通过构建的SLC35E1基因敲除小鼠动物模型研究银屑病或分枝杆菌感染性疾病,并验证SLC35E1基因在制备和筛选治疗银屑病或分枝杆菌感染性疾病药物中的应用,为人分枝杆菌感染性疾病的作用机制和治疗提供依据。
Description
技术领域
本发明涉及生物技术领域,具体涉及SLC35E1基因敲除小鼠动物模型的构建方法和应用。
背景技术
溶质转运载体35(Solute carrier 35,SLC35)家族,属于SLC家族中的第35个成员,分为7个亚型(A-G),是一种由300–400个氨基酸组成的疏水性蛋白质,属于Ⅲ型跨膜转运蛋白分子。SLC35多数成员定位在高尔基复合体膜上,另有一小部分定位在细胞膜上,主要转运细胞内相应单糖核苷酸并保证糖基化过程的顺利进行,SLC35被认为是糖基化途径中的关键调节因子。对SLC35缺陷型多细胞生物的生长发育研究及病理表型分析发现,SLC35转运蛋白缺乏可参与多种结缔组织病和遗传性疾病的发生,并且在肿瘤的发生及其转移等许多病理过程中发挥重要作用。
现有技术中,已知SLC35缺陷引起的疾病包括人类II型遗传性糖基化紊乱(CDGIIc)和II型遗传性包涵体肌病两种,近来还发现SLC35家族中某些成员与肿瘤的关系密切。此外,有研究显示在麻风患者中SLC35D1、SLC35D2的mRNA表达水平升高,提示SLC35D家族在麻风发病机制中可能具有重要作用,研究还发现SLC35E1在麻风病中发挥重要作用,通过对8个家系29个麻风患者及正常对照组进行全外显子测序,进一步扩大验证后发现麻风病患者研究中存在SLC35E1位点的突变;结合临床症状分析发现,SLC35E1是与皮损改变相关的易感基因。同时,SLC35E1的另一亚型SLC35E2B在斑块型银屑病患者的全外显子测序中被发现是突变基因,由此推断SLC35E1也可能参与银屑病的发病。
目前有关SLC35E1的研究甚少,其功能尚不清楚,SLC35E家族的成员SLC35E2B缺陷时可出现免疫缺陷及肌肉萎缩等症状,这与麻风病免疫功能紊乱及肌肉萎缩有相似临床症状,推测中国的麻风病患者发病可能与SLC35E2B基因突变有关。因此,建立敲除SLC35E1基因的C57小鼠模型,并在此基础上构建银屑病及分枝杆菌感染的模型对探究SLC35E1在银屑病及分枝杆菌感染中发挥的作用、相关机制及可能参与的分子通路具有重要意义,同时为指导银屑病和分枝杆菌感染性疾病的治疗提供新的理论依据。
发明内容
本发明的第一目的是提供SLC35E1基因敲除小鼠动物模型的构建方法。
本发明的第二个目的是提供SLC35E1基因在制备和筛选治疗银屑病或分枝杆菌感染性疾病药物中的应用。
为实现上述目的,本发明采用如下技术方案:
本发明提供的SLC35E1基因敲除小鼠动物模型的构建方法,基于CRISPR/Cas9基因敲除技术构建得到,包括以下步骤:
(1)根据SLC35E1基因序列确定SLC35E1小鼠待敲除基因的两对特异性靶点gRNA1和gRNA2、gRNA3和gRNA4,并与Cas9核酸酶体外转录成mRNA;
(2)将两对特异性靶点gRNA1和gRNA2、gRNA3和gRNA4和mRNA一起显微注射入小鼠受精卵内,显微注射后的受精卵移植至代孕母鼠输卵管内;
(3)待产出小鼠后继续传代培养,经基因型鉴定,得到稳定遗传的SLC35E1基因敲除小鼠动物模型;
其中,所述gRNA1的靶点序列如SEQ ID NO:1所示,所述gRNA2的靶点序列如SEQ IDNO:2所示,所述gRNA3的靶点序列如SEQ ID NO:3所示,所述gRNA4的靶点序列如SEQ ID NO:4所示。
优选地,步骤(3)中,显微注射后的受精卵移植至代孕母鼠输卵管内,产出小鼠,即为F0代小鼠;提取F0代小鼠尾部DNA,PCR扩增并将产物测序,将阳性小鼠与野生型异性小鼠交配获得F1代杂合子小鼠,将F1代杂合子小鼠杂交获得F2代纯合子小鼠,直至得到稳定遗传的SLC35E1基因敲除小鼠动物模型;其中,若为一个680bp的条带,为阳性纯合子;若为两个990bp和680bp的条带,为阳性杂合子;若为一个990bp的条带,为野生型对照。
本发明还提供一种SLC35E1基因敲除小鼠动物模型,通过上述SLC35E1基因敲除小鼠动物模型的构建方法得到。
本发明还提供SLC35E1基因敲除小鼠动物模型在银屑病或分枝杆菌感染性疾病研究中的应用。
本发明还提供SLC35E1基因在制备和筛选治疗银屑病药物中的应用。
本发明还提供SLC35E1基因在制备和筛选治疗分枝杆菌感染性疾病药物中的应用。
与现有技术相比,本发明的有益效果在于:
(1)本发明基于CRISPR/Cas9基因敲除技术设计SLC35E1小鼠待敲除基因的特异性靶点gRNA1、gRNA2、gRNA3和gRNA4,并成功构建SLC35E1基因敲除小鼠动物模型,经鉴定SLC35E1基因的exon1-4、exon2-5被敲除,为银屑病或分枝杆菌感染性疾病研究提供主要动物实验材料。
(2)本发明通过咪喹莫特乳膏构建SLC35E1基因缺陷小鼠银屑病模型、和用分枝杆菌感染构建SLC35E1基因缺陷小鼠感染模型,利用分子生物学、免疫组化染色、单细胞测序等手段研究SLC35E1基因在分枝杆菌感染过程中对银屑病、分枝杆菌感染性疾病的临床表现、病理特征、疾病状态、发病机制及可能的分子通路之间的关系,为研究SLC35E1家族在人分枝杆菌感染性疾病中的作用机制及相关治疗研究提供理论依据。
我们将参考以下详细说明更易于理解本发明的上述以及其他特征、方面和优点。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述,本发明的其它特征、目的和优点将会变得更显著:
图1是实施例中构建SLC35E1基因敲除小鼠动物模型的方法流程图。
图2是非目标gRNA分析引物;其中:(a)gRNA1的非目标分析序列;(b)gRNA2的非目标分析序列;(c)gRNA3的非目标分析序列;(d)gRNA4的非目标分析序列。
图3是咪喹莫特乳膏构建SLC35E1基因敲除小鼠银屑病模型的方法流程图。
图4是海鱼分枝杆菌构建SLC35E1基因敲除小鼠分枝杆菌感染模型的方法流程图。
图5是海鱼分枝杆菌构建的SLC35E1基因敲除小鼠分枝杆菌感染模型。
图6是实施例3中小鼠足垫脓液涂片的Ziehl-Neelsen抗酸染色。
图7是实施例3中小鼠足垫组织涂片的Ziehl-Neelsen抗酸染色。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例的附图,对本发明实施例的技术方案进行清楚、完整地描述。显然,所描述的实施例是本发明的一部分实施例,而不是全部的实施例。基于所描述的本发明的实施例,本领域普通技术人员在无需创造性劳动的前提下所获得的所有其它实施例,都属于本发明保护的范围。
以下实施例中敲除SLC35E1基因的基本信息为:敲除基因名称(Ensembl):ENSMUSG00000019731,敲除基因GenBank编码:NM_177766.3;敲除基因的exon数量:6,敲除针对的exon:exon1-4和exon2-5;构建的gRNA数目:2对;显微共注射:将体外转录生成的Cas9 mRNA和gRNA显微共注射到小鼠受精卵内。
实施例1
如图1和2所示,基于CRISPR/Cas9基因敲除技术构建SLC35E1基因敲除小鼠动物模型,步骤如下:
(1)根据SLC35E1基因序列(GenBank编码:NM_177766.3)确定SLC35E1小鼠待敲除基因的两对特异性靶点:
Pair1:gRNA1:aacgggtgagtagctacgcatgg(SEQ ID NO:1);
gRNA2:ggggatccacacataagtcctgg(SEQ ID NO:2);
Pair2:gRNA3:cgtgagaatccgacgcaccttgg(SEQ ID NO:3);
gRNA4:gtctacaagatatatgctcgagg(SEQ ID NO:4);
根据两对特异性靶点设计扩增引物序列如下:
SLC35E1-pair1-F:cattacagggccatttgaggagg(SEQ ID NO:5)
SLC35E1-pair1-R:gtgctgagtgaagcctggacctt(SEQ ID NO:6);
SLC35E1-pair2-F:ctgtgtctactatggtacgcgctgag(SEQ ID NO:7);
SLC35E1-pair2-R:gattcgcatcatacttggtctgtgtg(SEQ ID NO:8);
按照如表1所示的PCR反应体系及反应条件进行PCR扩增,与Cas9核酸酶体外转录成mRNA;
(2)将两对特异性靶点gRNA1和gRNA2、gRNA3和gRNA4和mRNA一起显微注射入小鼠受精卵内,显微注射后的受精卵移植至代孕母鼠输卵管内;
(3)显微注射后的受精卵移植至代孕母鼠输卵管内,产出小鼠,即为F0代小鼠;提取F0代小鼠尾部DNA,PCR扩增并将产物测序,将阳性小鼠与野生型异性小鼠交配获得F1代杂合子小鼠,将F1代杂合子小鼠杂交获得F2代纯合子小鼠,直至得到稳定遗传的SLC35E1基因敲除小鼠动物模型,其中按照如下基因鉴定引物进行基因型鉴定:
Mouse SLC35E1-F:ctgtgtctactatggtacgcgctgag(SEQ ID NO:9);
Mouse SLC35E1-R:gattcgcatcatacttggtctgtgtg(SEQ ID NO:10);
Mouse SLC35E1-Wt/He-F:atctgcccgagtatagatgtgccc(SEQ ID NO:11);
若为一个680bp的条带,为阳性纯合子;若为两个990bp和680bp的条带,为阳性杂合子;若为一个990bp的条带,为野生型对照,统计结果如表2所示。
表1
表2
实施例2
通过实施例1构建的SLC35E1基因敲除小鼠动物模型进一步构建银屑病动物模型验证SLC35E1基因在制备和筛选治疗银屑病药物中的应用。
按照如图3所示的方法,采用分子生物学、免疫组化染色和单细胞测序等手段研究SLC35E1基因与分枝杆菌感染过程中银屑病之间的关系。选取经基因型鉴定后的SLC35E1基因敲除小鼠,在SPF级环境中饲养至8周时,用咪喹莫特乳膏构建银屑病动物模型。
实施例3
通过实施例1构建的SLC35E1基因敲除小鼠动物模型进一步构建海鱼分枝杆菌感染动物模型验证SLC35E1基因在制备和筛选治疗分枝杆菌感染性疾病药物中的应用。
按照如图4所示的方法,采用分子生物学、免疫组化染色和单细胞测序等手段研究SLC35E1基因与分枝杆菌感染性疾病之间的关系。选取经基因型鉴定后的SLC35E1基因敲除小鼠,在SPF级环境中饲养至8周时,用海鱼分枝杆菌进行足垫的皮下注射构建分枝杆菌感染动物模型,如图5所示。
取上述小鼠足垫脓液进行涂片Ziehl-Neelsen抗酸染色,可见大量抗酸杆菌,如图6所示;取上述小鼠足垫组织及进行涂片Ziehl-Neelsen抗酸染色,可见抗酸杆菌如图7所示。
序列表
<110> 南方医科大学皮肤病医院(广东省皮肤病医院、广东省皮肤性病防治中心、中国麻风防治研究中心)
<120> SLC35E1基因敲除小鼠动物模型的构建方法和应用
<141> 2021-08-02
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 1
aacgggtgag tagctacgca tgg 23
<210> 2
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 2
ggggatccac acataagtcc tgg 23
<210> 3
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 3
cgtgagaatc cgacgcacct tgg 23
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 4
gtctacaaga tatatgctcg agg 23
<210> 5
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 5
cattacaggg ccatttgagg agg 23
<210> 6
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 6
gtgctgagtg aagcctggac ctt 23
<210> 7
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 7
ctgtgtctac tatggtacgc gctgag 26
<210> 8
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 8
gattcgcatc atacttggtc tgtgtg 26
<210> 9
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 9
ctgtgtctac tatggtacgc gctgag 26
<210> 10
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 10
gattcgcatc atacttggtc tgtgtg 26
<210> 11
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 11
atctgcccga gtatagatgt gccc 24
Claims (1)
1.SLC35E1基因敲除小鼠动物模型的构建方法,其特征在于,基于CRISPR/Cas9基因敲除技术构建,包括以下步骤:
(1)根据SLC35E1基因序列确定SLC35E1小鼠待敲除基因的两对特异性靶点gRNA1和gRNA2、gRNA3和gRNA4,并与Cas9核酸酶体外转录成mRNA,其中:
SLC35E1基因的GenBank编码为NM_177766.3;
所述gRNA1的靶点序列如SEQ ID NO:1所示,所述gRNA2的靶点序列如SEQ ID NO:2所示,所述gRNA3的靶点序列如SEQ ID NO:3所示,所述gRNA4的靶点序列如SEQ ID NO:4所示;
(2)将两对特异性靶点gRNA1和gRNA2、gRNA3和gRNA4和mRNA一起显微注射入小鼠受精卵内,显微注射后的受精卵移植至代孕母鼠输卵管内;
(3)显微注射后的受精卵移植至代孕母鼠输卵管内产出小鼠,为F0代小鼠;提取F0代小鼠尾部DNA,PCR扩增并将产物测序,阳性小鼠与野生型异性小鼠交配获得F1代阳性杂合子小鼠,F1代阳性杂合子小鼠杂交获得F2代阳性纯合子小鼠,经基因型鉴定:
若为一个680bp的条带,为阳性纯合子;若为两个990bp和680bp的条带,为阳性杂合子;若为一个990bp的条带,为野生型对照,得到稳定遗传的SLC35E1基因敲除小鼠动物模型。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2021110825617 | 2021-09-15 | ||
CN202111082561 | 2021-09-15 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114410691A CN114410691A (zh) | 2022-04-29 |
CN114410691B true CN114410691B (zh) | 2022-09-30 |
Family
ID=81262051
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210168465.2A Active CN114410691B (zh) | 2021-09-15 | 2022-02-23 | Slc35e1基因敲除小鼠动物模型的构建方法和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114410691B (zh) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011014721A2 (en) * | 2009-07-30 | 2011-02-03 | Transposagen Biopharmaceuticals, Inc. | Genetically modified rat models for pharmacokinetics |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3307888A1 (en) * | 2015-06-12 | 2018-04-18 | Erasmus University Medical Center Rotterdam | New crispr assays |
-
2022
- 2022-02-23 CN CN202210168465.2A patent/CN114410691B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011014721A2 (en) * | 2009-07-30 | 2011-02-03 | Transposagen Biopharmaceuticals, Inc. | Genetically modified rat models for pharmacokinetics |
Non-Patent Citations (2)
Title |
---|
Identification and analysis of genes from the mouse otic vesicle and their association with developmental subprocesses through in situ hybridization;NicolaPowles 等;《Developmental Biology》;20040401;第268卷(第1期);第24-38页 * |
溶质转运蛋白(SLC)与皮肤病;郑宝庆 等;《中国皮肤性病学杂志》;20190630;第33卷(第6期);第709-712页 * |
Also Published As
Publication number | Publication date |
---|---|
CN114410691A (zh) | 2022-04-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107858373B (zh) | 内皮细胞条件性敲除ccr5基因小鼠模型的构建方法 | |
WO2018177351A1 (zh) | 基于CRISPR/Cas9技术的制备无嵌合基因敲除动物的方法 | |
CN106929533B (zh) | Kars基因点突变小鼠模型的构建方法及其应用 | |
CN104593418A (zh) | 一种人源化大鼠药物评价动物模型建立的方法 | |
CN106282231B (zh) | 粘多糖贮积症ii型动物模型的构建方法及应用 | |
CN110257435B (zh) | 一种prom1-ko小鼠模型的构建方法及其应用 | |
JP2010029219A (ja) | 動物モデルの開発のための方法 | |
CN111690689B (zh) | 人源化ccr2基因改造动物模型的构建方法及其应用 | |
CN110438160B (zh) | 一种Cd2ap基因敲除动物的构建方法及应用 | |
CN110643716A (zh) | 一种与绵羊尾脂重相关的分子标记及其应用 | |
CN113699152A (zh) | Slc35e2b基因敲除小鼠动物模型的构建方法和应用 | |
CN109207524A (zh) | 基于fto基因的人类肥胖症斑马鱼模型的建立与应用 | |
CN114657264B (zh) | 一种胡子鲇性别特异性分子标记引物及其应用 | |
CN114774413A (zh) | 斑马鱼排卵障碍模型的构建方法、检测方法及应用 | |
CN111560401A (zh) | 一种翘嘴红鲌和团头鲂肌间刺变粗的分子育种方法 | |
CN109929875B (zh) | 一种lag3基因人源化动物模型的构建方法及其应用 | |
CN117604034A (zh) | 一种制备人源化ttr小鼠的方法及其应用 | |
CN110115248A (zh) | 一种生长抑素基因缺陷的免疫缺陷小鼠、其制备方法及其用途 | |
CN114410691B (zh) | Slc35e1基因敲除小鼠动物模型的构建方法和应用 | |
CN116694756A (zh) | 家族性成人肌阵挛癫痫的诊断标志物和应用 | |
CN114480497B (zh) | 一种ep400基因敲除斑马鱼心力衰竭模型的构建及其应用的方法 | |
CN112626122B (zh) | hKDR人源化小鼠模型及其建立方法和应用 | |
CN109694885B (zh) | 基于CRISPR/Cas9技术制备PI3Kγ全身敲除模式小鼠方法及其应用和试剂盒 | |
CN111972355A (zh) | GSDIa型糖原累积症小鼠模型及其构建方法 | |
CN110592122A (zh) | 斑马鱼naalad2基因启动子及其应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |