CN112877357A - Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途 - Google Patents
Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途 Download PDFInfo
- Publication number
- CN112877357A CN112877357A CN202011607045.7A CN202011607045A CN112877357A CN 112877357 A CN112877357 A CN 112877357A CN 202011607045 A CN202011607045 A CN 202011607045A CN 112877357 A CN112877357 A CN 112877357A
- Authority
- CN
- China
- Prior art keywords
- mouse
- fragment
- obesity
- gene
- abcg4
- 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
- 101150071818 Abcg4 gene Proteins 0.000 title claims abstract description 43
- 208000008589 Obesity Diseases 0.000 title claims abstract description 36
- 235000020824 obesity Nutrition 0.000 title claims abstract description 36
- 238000010171 animal model Methods 0.000 title claims abstract description 20
- 230000002457 bidirectional effect Effects 0.000 title claims description 7
- 230000008859 change Effects 0.000 title abstract description 8
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 claims abstract description 11
- 230000014509 gene expression Effects 0.000 claims abstract description 9
- 241001465754 Metazoa Species 0.000 claims abstract description 7
- 238000012216 screening Methods 0.000 claims abstract description 7
- 210000004369 blood Anatomy 0.000 claims abstract description 6
- 239000008280 blood Substances 0.000 claims abstract description 6
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 38
- 239000012634 fragment Substances 0.000 claims description 34
- 210000004027 cell Anatomy 0.000 claims description 16
- 230000008685 targeting Effects 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 13
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 claims description 6
- 210000002459 blastocyst Anatomy 0.000 claims description 6
- 238000010367 cloning Methods 0.000 claims description 6
- 230000004927 fusion Effects 0.000 claims description 6
- 238000002744 homologous recombination Methods 0.000 claims description 6
- 230000006801 homologous recombination Effects 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 241000124008 Mammalia Species 0.000 claims description 5
- 108700024394 Exon Proteins 0.000 claims description 4
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 4
- 108091026890 Coding region Proteins 0.000 claims description 3
- 101100489898 Mus musculus Abcg4 gene Proteins 0.000 claims description 3
- 230000005540 biological transmission Effects 0.000 claims description 3
- 230000007547 defect Effects 0.000 claims description 3
- 210000004602 germ cell Anatomy 0.000 claims description 3
- 230000006798 recombination Effects 0.000 claims description 2
- 238000005215 recombination Methods 0.000 claims description 2
- 201000010099 disease Diseases 0.000 abstract description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 9
- 239000003814 drug Substances 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 3
- 230000001419 dependent effect Effects 0.000 abstract description 2
- 238000012239 gene modification Methods 0.000 abstract description 2
- 230000005017 genetic modification Effects 0.000 abstract description 2
- 235000013617 genetically modified food Nutrition 0.000 abstract description 2
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 241000699670 Mus sp. Species 0.000 description 25
- 108090000623 proteins and genes Proteins 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 10
- 230000002950 deficient Effects 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 9
- 210000000577 adipose tissue Anatomy 0.000 description 8
- 210000004556 brain Anatomy 0.000 description 8
- 238000003752 polymerase chain reaction Methods 0.000 description 8
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 6
- 229930186217 Glycolipid Natural products 0.000 description 6
- 238000002105 Southern blotting Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 230000004060 metabolic process Effects 0.000 description 6
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000037396 body weight Effects 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 3
- 238000003209 gene knockout Methods 0.000 description 3
- 210000000496 pancreas Anatomy 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000003633 gene expression assay Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 101150033839 4 gene Proteins 0.000 description 1
- 101150048714 75 gene Proteins 0.000 description 1
- 108010006533 ATP-Binding Cassette Transporters Proteins 0.000 description 1
- 102000005416 ATP-Binding Cassette Transporters Human genes 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 101000583080 Bunodosoma granuliferum Delta-actitoxin-Bgr2a Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 description 1
- 208000004930 Fatty Liver Diseases 0.000 description 1
- 206010019708 Hepatic steatosis Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010033307 Overweight Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 238000012098 association analyses Methods 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 description 1
- 229960005156 digoxin Drugs 0.000 description 1
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 235000020774 essential nutrients Nutrition 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 208000010706 fatty liver disease Diseases 0.000 description 1
- IRSCQMHQWWYFCW-UHFFFAOYSA-N ganciclovir Chemical compound O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 description 1
- 229960002963 ganciclovir Drugs 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 238000003205 genotyping method Methods 0.000 description 1
- 238000004442 gravimetric analysis Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000011623 obesity animal model Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
-
- 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
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
- A01K2217/054—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function
-
- 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
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
-
- 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
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Plant Pathology (AREA)
- Immunology (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明属于动物基因工程和遗传修饰领域,具体地说,涉及Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途。所述用途具体是指通过敲除动物体内Abcg4基因,或抑制Abcg4基因的表达,来构建动物模型。所述动物模型的肥胖程度依赖于性别,表现为以同窝生同性别野生型为参照,雌性变胖且血液甘油三酯含量降低,雄性变瘦且血液甘油三酯含量升高。本发明的模型可用于研究肥胖症及相关疾病,并可用于筛选出更加有效、更有针对性的靶向治疗肥胖或治疗血浆甘油三酯升高的药物。
Description
技术领域
本发明属于动物基因工程和遗传修饰领域,具体的说,涉及Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途。
背景技术
糖和脂质是人体必需的营养物质,它们既为细胞提供能量同时也是细胞的基本结构成分。糖脂代谢在健康的个体中能被精准地调节,糖脂代谢异常则会引起包括肥胖症、脂肪肝、糖尿病、动脉粥样硬化、心脑血管疾病,甚至癌症等许多疾病。因此,糖脂代谢稳态平衡对机体的健康起着重要的作用。
肥胖是由身体的脂肪过量累积所产生的一种复杂的疾病,而肥胖基因,是指基因所编码蛋白质是一种食欲与能量平衡调节途径的组成部分,这种途径的失衡直接或者间接导致体脂肪的积累和体重增加。肥胖不仅仅影响人体的美观,更可以增加其它疾病和健康问题的患病风险。在北美洲,三分之二的美国成年人被归为超重或肥胖。
肥胖受遗传因素即基因的影响,近年来,大规模的基因组关联研究(GWA Ss,genome-wide association studies)方法应用于肥胖相关基因的鉴定,但一般找出的基因数量比较多,单个基因分析时其对肥胖的影响并不大,不同实验室的结果也不尽一致,能相互验证的例子较少。比如Fall T等运用GWAS和荟萃分析(meta-analyses)发现有超过75个基因位点与肥胖相关。最近,另有研究报道,通过全基因组关联分析发现了500多个与肥胖特征相关的基因位点,但每一个位点内精确定位致病基因仍很困难。
疾病动物模型可以为了解疾病发生发展机制,发展疾病预防和干预方法提供巨大帮助。作为哺乳动物,小鼠遗传操作和表型分析技术最为成熟,在各种疾病动物模型培育研究中占主导地位。但长期以来所使用的肥胖动物模型在不同的性别间其肥胖程度没有差异,且至今尚无不同性别间肥胖程度呈双向变化的动物模型的相关报道。
发明内容
为了克服现有技术中存在的问题,本发明的目的在于提供Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途。
为了实现上述目的,本发明采用如下技术方案:
本发明的第一个方面,提供Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途。
进一步的,所述用途具体是指通过敲除动物体内Abcg4基因或抑制Abcg4基因的表达,来构建动物模型。
更进一步的,所述动物模型的肥胖程度依赖于性别,表现为以同窝生同性别野生型为参照,雌性变胖且血液甘油三酯含量降低,雄性变瘦且血液甘油三酯含量升高。
更进一步的,所述动物为哺乳动物。
更进一步的,所述哺乳动物为小鼠。
更进一步的,敲除小鼠Abcg4基因的方法包括如下步骤:
S1、以野生型小鼠基因组为模板,以引物对I扩增包含Abcg4基因第6外显子至部分第7外显子的片段,记作片段I;
所述引物对I的核苷酸序列为:
FI:5’-TGGTGGATGGTGCAGATGAC-3’;
RI:5’-AAATCGGGGTGGTGCTTAGG3’;
以引物对II扩增包含Abcg4基因第9-14外显子的片段,记作片段II;
所述引物对II的核苷酸序列为:
FII:5’-AACTGACGAAGAAGCCGGAG3’;
RII:5’-ATCAAAGAAGGACTGCCCCG3’;
S2、将片段I插入到pCRII载体的eGFP编码区前,获得融合片段I,将融合片段I插入到靶向载体OSDUPDEL的多克隆位点A,将片段II插入到同一个靶向载体OSDUPDEL的多克隆位点B,获得靶向重组载体;
S3,上述重组载体经注射胚胎干细胞进行同源重组后,获得ES细胞打靶产物,对ES细胞打靶产物进行阳性克隆鉴定,筛选出正向敲除的ES细胞;
S4,将S3筛选出的ES细胞注射到C57BL/6J小鼠囊胚中并植入假孕母鼠体内,获得具有种系传递能力的杂合子代小鼠;
S5,将上述杂合子代小鼠与C57BL/6J小鼠进行N代的回交,产生Abcg4基因重要功能部位缺陷鼠种系,杂合子相互交配,生成Abcg4基因缺陷型小鼠。
更进一步的,S5中,N≥16。
与现有技术相比,本发明具有如下有益效果:
1、本发明将基因敲除的小鼠用作肥胖或肥胖相关疾病的性别间双向变化的特异动物模型,主要通过减少或完全敲除小鼠的至少一种组织或至少一种细胞类型中的ATP结合盒转运蛋白超家族G亚族中第4号成员基因(简称Abcg4基因)的量的方法来制作。
2、现有技术所使用的肥胖动物模型在不同的性别间其肥胖程度没有差异,本发明所发现的性别间肥胖的反向变化,更适合于肥胖及其相关药物的个性化研究与开发。
3、在肥胖及其糖尿病等相关疾病的研究上,可以使用该动物模型筛选出更加有效、更有针对性的靶向治疗肥胖的药物,也可以利用其筛选出可以靶向降低血浆中甘油三酯的药物。
附图说明
图1为实施例1构建Abcg4基因重要功能域缺失鼠的策略示意图。
图2为实施例2用Southern杂交证实小鼠同源重组的结果。
图3为Abcg4基因缺陷型小鼠和野生型小鼠甘油三酯含量比较。
图4为Abcg4基因缺陷型小鼠和野生型小鼠性腺旁脂肪垫重量比较(图A和图B为同一个实验的不同画法,图B中连线表示同窝生)。
图5为Abcg4基因缺陷型小鼠和野生型小鼠含脂率比较(图A和图B为同一个实验的不同画法,图B中连线表示同窝生)。
图6为Abcg4基因缺陷型小鼠和野生型小鼠胰腺重量比较(图A和图B为同一个实验的不同画法,图B中连线表示同窝生)。
图7为Abcg4基因敲除雌鼠鼠脑实时定量PCR基因表达测定。
图8为Abcg4基因敲除雄鼠脑实时定量PCR基因表达测定。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明,但不应理解为本发明的限制。如未特殊说明,下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段,下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1
参见图1,本实施例的敲除小鼠Abcg4基因的方法,包括如下步骤:
以野生型小鼠基因组为模板,以引物对I扩增包含Abcg4基因第6外显子至部分第7外显子的片段,记作片段I;
所述引物对I的核苷酸序列为:
FI:5’-TGGTGGATGGTGCAGATGAC-3’(如SEQ ID NO.1所示);
RI:5’-AAATCGGGGTGGTGCTTAGG-3’(如SEQ ID NO.2所示);
以引物对II扩增包含Abcg4基因第9-14外显子的片段,记作片段II;
所述引物对II的核苷酸序列为:
FII:5’-AACTGACGAAGAAGCCGGAG-3’(如SEQ ID NO.3所示);
RII:5’-ATCAAAGAAGGACTGCCCCG-3’(如SEQ ID NO.4所示);
以引物对II扩增包含Abcg4基因第9-14外显子的片段,记作片段II;
S2、将片段I插入到pCRII载体的eGFP编码区前,获得融合片段I,将融合片段I插入到靶向载体OSDUPDEL(OSDupDel Gene Targeting Vector-neo)载体的多克隆位点A(MCSA),将片段II插入到同一个靶向载体OSDUPDEL载体的多克隆位点B(MCSB),获得靶向重组载体;
上述PCR扩增利用Roche的“扩展长模板PCR系统”试剂盒(Expanded LongTemplate PCR System kit,Roche Applied Science,Indianapolis,IA,USA)。
程序为:94℃初始变性3分钟;先以94℃15秒,65℃30秒,68℃8分钟,进行10个循环,然后以94℃15秒,65℃30秒,68℃8分20秒进行20个循环;最终68℃延伸7分钟。
S3,上述靶向重组载体经注射胚胎干细胞进行同源重组后,获得ES细胞打靶产物,对ES细胞打靶产物进行阳性克隆鉴定,筛选出正向敲除的ES细胞;
阳性ES细胞基因组鉴定方法:将靶向重组载体电转到C57B6/J小鼠胚胎干细胞中,转染细胞用G418作正向(阳性)选择,用更昔洛韦作负向(阴性)选择,存活的克隆通过PCR和Southern印迹法进行筛选;
S4,将S3筛选出的ES细胞注射到C57BL/6J小鼠囊胚中并植入假孕母鼠体内,获得具有种系传递能力的杂合子代小鼠;
S5,将上述杂合子代小鼠与C57BL/6J小鼠进行16代的回交,产生Abcg4基因重要功能部位缺陷鼠种系,杂合子相互交配,生成Abcg4基因缺陷型小鼠。
实施例2
Abcg4基因缺陷型小鼠基因型鉴定
用Southern杂交证实同源重组的正确性,包括如下步骤:
先用BgII酶消化实施例1获得的Abcg4基因缺陷型小鼠的基因组DNA以及野生型小鼠基因组DNA,制作探针进行Southern杂交检测。
所述探针的制备方法如下:先用引物int2F-2和int5R对基因组DNA进行PCR扩增。int2F-2和int5R序列信息如下:
int2F:5’-CAGTACACAGGTTAAGCTAGGCAG-3’(如SEQ ID NO.5所示);
int5R:5’-TCCATACACAGGCCTCGCCTAAGC-3’(如SEQ ID NO.6所示)。
PCR反应体系为:DNA模版10ng,引物各2ug,Taq酶1unit,1×PCR buffer,反应总体积为20ul。
PCR反应条件:94℃,5分钟;循环:94℃,30秒,60℃,45秒,72℃,2分钟,重复30个循环;最后:72℃,10分钟;然后于4℃保存。PCR产物琼脂糖凝胶电泳并切胶纯化后,用罗氏的地高辛标记试剂盒(DIG-High Prime DNA Labeling and Detection System)进行标记,纯化后作为探针用于Southern杂交。
结果如图2所示,具有靶向插入外源片段的靶片段(6.4kb)大于野生型(WT)片段(4.5kb)。基因敲除鼠父母双方均为杂合子,使其交配所产生的子代中,Abcg4敲除的纯合子(ko)Southern杂交的结果仅有6.4kb大小的一条带(右边数起第3个泳道),野生型(wt)小鼠仅含一条约4.5kb的一条带(最右边),其它两个小鼠为杂合型(6.4kb和4.5kb的带子同时存在)。此实验证明,通过基因组的靶向同源重组所产生的Abcg4基因重要功能域敲除的小鼠后代,其基因型及Southern杂交的片段大小与理论值相符,证明了靶向敲除的正确性。(注:最左边的分子量标记位置用溴化乙锭进行凝胶染色来估算。)
实施例3
利用肥胖症指标表征Abcg4基因缺陷型小鼠
以下结果均是以出生后4周同窝生Abcg4基因缺陷型小鼠和野生型小鼠进行的比较。
(1)甘油三酯含量分析
分析过程及结果:使用Boehringer Mannheim公司的甘油三酯分析试剂盒,根据其说明书操作,结果如图3所示。
(2)性腺旁脂肪垫分析
分析过程及结果:小鼠麻醉,称取并记录体重,解剖小鼠取出性腺旁脂肪垫,称重并记录,结果如图4所示。脂肪垫重量与该小鼠的体重之比即为含脂率(以百分比计),结果如图5所示。
(3)胰腺重量分析
分析过程及结果:解剖小鼠取出胰腺,并记录,胰腺重量结果如图6所示。
实施例4
利用小鼠脑内糖脂代谢相关基因的表达,从分子水平侧面验证了Abcg4基因缺陷型小鼠在其脂肪垫/体重百分比变化的性别“二态性”
(1)Abcg4基因敲除鼠的雌性鼠与同窝生的野生型雌鼠相比,其脑内与糖脂代谢相关的系列基因的表达情况,以实验当时连接实时定量PCR仪的电脑软件直接输出的结果(print screen)复制而来,代表没有对图片进行任何人为的加工与修饰。野生型小鼠基因的相对表达量设为1,如图7所示。
(2)基因敲除雄鼠与同窝生野生型小鼠相比,脑内的糖脂代谢相关基因表达情况如图8所示。
从以上不同性别Abcg4基因敲除小鼠脑内基因表达结果可以看出,雌性与雄性基因敲除鼠高表达与低表达的基因几乎是相反的。原来基因敲除雌鼠脑内基因表达相对较低的几个基因在雄鼠脑内其表达明显升高。这与本实验观察到的雌性小鼠性腺旁脂肪垫重量与体重之比率比野生型小鼠增加(变胖),而雄鼠该比例降低(变瘦)的表型相吻合。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若针对本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
序列表
<110> 江苏科技大学
<>
<120> Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途
<>
<160> 6
<>
<170> SIPOSequenceListing 1.0
<>
<210> 1
<211> 20
<212> DNA
<213> 人工序列
<>
<400> 1
tggtggatgg tgcagatgac 20 <>
<>
<210> 2
<211> 20
<212> DNA
<213> 人工序列
<>
<400> 2
aaatcggggt ggtgcttagg 20 <>
<>
<210> 3
<211> 20
<212> DNA
<213> 人工序列
<>
<400> 3
aactgacgaa gaagccggag 20 <>
<>
<210> 4
<211> 20
<212> DNA
<213> 人工序列
<>
<400> 4
atcaaagaag gactgccccg 20 <>
<>
<210> 5
<211> 24
<212> DNA
<213> 人工序列
<>
<400> 5
cagtacacag gttaagctag gcag 24 <>
<>
<210> 6
<211> 24
<212> DNA
<213> 人工序列
<>
<400> 6
tccatacaca ggcctcgcct aagc 24 <>
<>
<>
<>
Claims (7)
1.Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途。
2.根据权利要求1所述的用途,其特征在于,所述用途具体是指通过敲除动物体内Abcg4基因或抑制Abcg4基因的表达,来构建动物模型。
3.根据权利要求2所述的用途,其特征在于,所述动物模型的肥胖程度依赖于性别,表现为以同窝生同性别野生型为参照,雌性变胖且血液甘油三酯含量降低,雄性变瘦且血液甘油三酯含量升高。
4.根据权利要求3所述的用途,其特征在于,所述动物为哺乳动物。
5.根据权利要求4所述的用途,其特征在于,所述哺乳动物为小鼠。
6.根据权利要求5所述的用途,其特征在于,敲除小鼠Abcg4基因的方法包括如下步骤:
S1、以野生型小鼠基因组为模板,以引物对I扩增包含Abcg4基因第6外显子至部分第7外显子的片段,记作片段I;
所述引物对I的核苷酸序列为:
FI:5’-TGGTGGATGGTGCAGATGAC-3’;
RI:5’-AAATCGGGGTGGTGCTTAGG3’;
以引物对II扩增包含Abcg4基因第9-14外显子的片段,记作片段II;
所述引物对II的核苷酸序列为:
FII:5’-AACTGACGAAGAAGCCGGAG3’;
RII:5’-ATCAAAGAAGGACTGCCCCG3’;
S2、将片段I插入到pCRII载体的eGFP编码区前,获得融合片段I,将融合片段I插入到靶向载体OSDUPDEL的多克隆位点A,将片段II插入到同一个靶向载体OSDUPDEL的多克隆位点B,获得靶向重组载体;
S3,上述重组载体经注射胚胎干细胞进行同源重组后,获得ES细胞打靶产物,对ES细胞打靶产物进行阳性克隆鉴定,筛选出正向敲除的ES细胞;
S4,将S3筛选出的ES细胞注射到C57BL/6J小鼠囊胚中并植入假孕母鼠体内,获得具有种系传递能力的杂合子代小鼠;
S5,将上述杂合子代小鼠与C57BL/6J小鼠进行N代的回交,产生Abcg4基因重要功能部位缺陷鼠种系,杂合子相互交配,生成Abcg4基因缺陷型小鼠。
7.根据权利要求6所述的用途,其特征在于,S5中,N≥16。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011607045.7A CN112877357B (zh) | 2020-12-28 | 2020-12-28 | Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途 |
GB2208633.4A GB2616924A (en) | 2020-12-28 | 2021-05-25 | Use of ABCG4 gene in construction of animal model having bidirectionally changed obesity degree |
PCT/CN2021/095776 WO2022142092A1 (zh) | 2020-12-28 | 2021-05-25 | Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202011607045.7A CN112877357B (zh) | 2020-12-28 | 2020-12-28 | Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112877357A true CN112877357A (zh) | 2021-06-01 |
CN112877357B CN112877357B (zh) | 2021-12-24 |
Family
ID=76046347
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202011607045.7A Active CN112877357B (zh) | 2020-12-28 | 2020-12-28 | Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途 |
Country Status (3)
Country | Link |
---|---|
CN (1) | CN112877357B (zh) |
GB (1) | GB2616924A (zh) |
WO (1) | WO2022142092A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117210497A (zh) * | 2023-08-16 | 2023-12-12 | 江苏科技大学 | Abcg4基因在构建脑动脉粥样硬化伴肺部增大小鼠模型中的应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002070691A2 (en) * | 2001-03-02 | 2002-09-12 | Active Pass Pharmaceuticals, Inc. | Abcg4 transporter and uses thereof |
CN102191274A (zh) * | 2011-04-07 | 2011-09-21 | 江苏科技大学 | 阿尔茨海默病动物模型的制备方法 |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103952412A (zh) * | 2014-05-06 | 2014-07-30 | 江苏科技大学 | 小鼠脑Abcg4基因沉默对AD相关基因表达影响的实验方法 |
WO2020169472A2 (en) * | 2019-02-18 | 2020-08-27 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods of inducing phenotypic changes in macrophages |
-
2020
- 2020-12-28 CN CN202011607045.7A patent/CN112877357B/zh active Active
-
2021
- 2021-05-25 GB GB2208633.4A patent/GB2616924A/en active Pending
- 2021-05-25 WO PCT/CN2021/095776 patent/WO2022142092A1/zh active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002070691A2 (en) * | 2001-03-02 | 2002-09-12 | Active Pass Pharmaceuticals, Inc. | Abcg4 transporter and uses thereof |
CN102191274A (zh) * | 2011-04-07 | 2011-09-21 | 江苏科技大学 | 阿尔茨海默病动物模型的制备方法 |
Non-Patent Citations (2)
Title |
---|
WU JINMEI等: "Gender-specific effect of deficient Abcg4 on lipogenesis in mouse brain", 《南京大学学报(自然科学)》 * |
WU JINMEI等: "Opposite effects of Abcg4 knockout on gonadal fat/body weight ratio changes between male and female mouse littermates", 《JOURNAL OF INVESTIGATIVE MEDICINE》 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN117210497A (zh) * | 2023-08-16 | 2023-12-12 | 江苏科技大学 | Abcg4基因在构建脑动脉粥样硬化伴肺部增大小鼠模型中的应用 |
Also Published As
Publication number | Publication date |
---|---|
GB202208633D0 (en) | 2022-07-27 |
CN112877357B (zh) | 2021-12-24 |
WO2022142092A1 (zh) | 2022-07-07 |
GB2616924A (en) | 2023-09-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Skryabin et al. | Deletion of the MBII-85 snoRNA gene cluster in mice results in postnatal growth retardation | |
WO2018177351A1 (zh) | 基于CRISPR/Cas9技术的制备无嵌合基因敲除动物的方法 | |
CN110885858B (zh) | Amy1基因敲除小鼠动物模型的构建方法及应用 | |
CN110771573B (zh) | PirB基因敲入的小鼠动物模型及其构建方法 | |
CN110804629A (zh) | PirB基因敲除小鼠动物模型及其构建方法 | |
Wang et al. | Generation of gene-knockout Mongolian gerbils via CRISPR/Cas9 system | |
CN112877357B (zh) | Abcg4基因在构建肥胖程度呈双向变化动物模型中的用途 | |
JPWO2003004637A1 (ja) | 体重または摂食量の調節に関与するヒスタミン受容体h3遺伝子の利用 | |
PENTZ et al. | Ren1d and Ren2 cooperate to preserve homeostasis: evidence from mice expressing GFP in place of Ren1d | |
Monecke et al. | Duper: A mutation that shortens hamster circadian period | |
CN114107382A (zh) | G3bp1条件性基因敲除小鼠模型 | |
CN117604034A (zh) | 一种制备人源化ttr小鼠的方法及其应用 | |
CN113897369A (zh) | KRT10定点基因敲入P2A-CrePR1-T2A-tdTomato小鼠模型的构建及应用 | |
WO2024103631A1 (zh) | Notch2nlc基因ggc重复扩增突变转基因小鼠及其构建方法和应用 | |
CN114591957B (zh) | 一种重症a型血友病动物模型的构建方法及其应用 | |
EP2882284A1 (en) | Animal model of krabbe's disease | |
CN116716345A (zh) | 一种用于制备人源化rho和或rho突变体小鼠的方法及其应用 | |
CN114134152B (zh) | Glp1r基因人源化的非人动物及其构建方法和应用 | |
CN115044619B (zh) | 一种全身诱导性Ppp3ca基因敲除鼠模型的构建方法 | |
CN114457114B (zh) | 一种Fars2基因条件性敲除动物模型的构建方法 | |
WO1993019166A1 (en) | Small animal model for studying cholesterol metabolism | |
Fan et al. | The Hepatocyte nuclear factor-1 alpha (HNF1A) gene is associated with fatness and loin muscle area in the pig | |
Mérillat et al. | Conditional gene targeting of the ENaC subunit genes Scnn1b and Scnn1g | |
EP1934594A2 (en) | An animal model for studying atherosclerotic lesions | |
CN117363660B (zh) | 一种构建sma小鼠模型的方法 |
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 |