WO2023216748A1 - 一种FTO rs1421085 T＞C点突变小鼠模型及其应用 - Google Patents

一种FTO rs1421085 T＞C点突变小鼠模型及其应用 Download PDF

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WO2023216748A1
WO2023216748A1 PCT/CN2023/084657 CN2023084657W WO2023216748A1 WO 2023216748 A1 WO2023216748 A1 WO 2023216748A1 CN 2023084657 W CN2023084657 W CN 2023084657W WO 2023216748 A1 WO2023216748 A1 WO 2023216748A1
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fto
mouse model
point mutation
mice
fat
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宁光
王计秋
刘瑞欣
洪洁
张织茵
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上海交通大学医学院附属瑞金医院
上海市内分泌代谢病研究所
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • 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

Definitions

  • the invention belongs to the field of animal models, and particularly relates to a FTO rs1421085 T>C point mutation mouse model and its application.
  • leptin receptor leptin receptor
  • melanocortin receptor melanocortin receptor
  • obesity monogenic obesity
  • genetics alone cannot fully explain the rapid expansion of obesity worldwide in the past 30 years, it may explain the reasons for weight differences among individuals in the same environment. That is, the relative position of an individual in the overall population weight curve is mainly It is determined by genetic factors, whether at a lower level or a higher level of socioeconomic development; at the same time, the discovery of obesity susceptibility genes can deepen our understanding of the pathophysiological mechanisms of body weight regulation and fat distribution differences, and provide insights into the treatment of obesity. and provide new ideas for prevention.
  • the technical problem to be solved by this invention is to provide a FTO rs1421085 T>C point mutation mouse model and its application.
  • An FTO rs1421085 T>C point mutation mouse model of the present invention is used in studying obesity diseases.
  • the applications include, but are not limited to, drug screening and diagnostic target discovery.
  • An FTO rs1421085 T>C point mutation mouse model of the present invention is used in screening or preparing anti-obesity drugs.
  • the mouse model is a CRISPR/Cas-based FTO rs1421085 T>C point mutation mouse model, an ES-based One or more of the target FTO rs1421085 T>C point mutation mouse model and the brown fat conditional FTO rs1421085 T>C point mutation mouse model.
  • the construction method of the CRISPR/Cas-based FTO rs1421085 T>C point mutation mouse model is: construct a donor gene sequence containing rs1421085, and introduce it together with Cas9 mRNA and sgRNA through microinjection for fertilization in vitro In ovo, it was subsequently injected into surrogate female mice to identify the genotype and obtain the FTO rs1421085 T>C point mutation mouse model.
  • the construction method of the FTO rs1421085 T>C point mutation mouse model based on ES targeting is: construct a donor gene sequence containing rs1421085, perform DNA homologous recombination in mouse embryonic stem cells ES cells, and re-inject the ES cells Chimeric embryos were formed in the blastocoel, developed in pseudopregnant female mice, and the genotype was identified to obtain the FTO rs1421085 T>C point mutation mouse model.
  • the Cre/Loxp system constructs a method for constructing a brown fat conditional FTO rs1421085 T>C point mutation mouse model: select the insertion site: use the UCSC online database to search upstream and downstream of the SNP (rs1421085) near the SNP (conditional mutation region ⁇ 3kb) non-conserved region. A non-conserved region was found 2.6kb upstream of the SNP, which served as the insertion site for 1st loxP; a non-conserved region was found 0.3kb downstream, which served as the insertion site for 2ndloxP.
  • the partial region of intron 1 containing the SNP rs1421085 was reverse cloned to the upstream of exon 2; this region is flanked by loxP and lox2272 sites respectively; ES cells of C57BL/6 strain mice The target cell into which the targeting vector will be inserted.
  • the present invention provides the FTO rs1421085 T>C point mutation mouse model and construction method for the first time. Moreover, the FTO rs1421085 T>C point mutation mouse model constructed by the present invention provides an effective experimental animal model for screening or preparing anti-obesity drugs, and has good application prospects.
  • Figure 1 is a schematic diagram of the principle of CRISPR/Cas technology to construct FTO rs1421085 T>C point mutation mice;
  • Figure 2 shows the DNA sequencing results of wild-type mice (top) and HE mice (bottom);
  • Figure 3 shows that compared with the control, male rs1421085 mutant mice have improved metabolism and can better resist HFD-induced obesity;
  • EF Insulin tolerance test
  • F area under the curve
  • n 17.
  • Figure 4 shows that ES male rs1421085 T>C systemic mutant mice have enhanced heat production capacity of brown adipose tissue compared with the control group;
  • Figure 5 shows the resistance of rs1421085 systemic mutant mice to high-fat-dependent classic BAT;
  • A Timeline of iBAT resection and HFD feeding.
  • B Gross photo of mice after iBAT resection and sacrificed at 24 weeks of age.
  • Figure 6 shows the construction of UKI (Ucp1-Cre; rs1421085 Knock-in) mice
  • Figure 7 shows the resistance of male UKI mice to high-fat-induced obesity
  • E Photographs of HE staining of iBAT and iWAT. The scale shown in the picture is 200um.
  • F BAT electron microscope photo, the scale bar in the picture is 1um, and the red arrow indicates mitochondria.
  • (G) q-PCR detection of mRNA expression levels of thermogenesis and differentiation-related genes in BAT; n 14:9.
  • (H) The left picture shows Western-blot detection of Ucp1, Pgc1- ⁇ , Ppar- ⁇ , and C/ebp ⁇ protein expression levels in BAT, and the right picture shows the quantification of relative protein expression levels (normalized by Hsp90 protein expression level); n 3:3. All data are expressed as mean ⁇ SEM, and two-tailed t test was used to calculate statistical differences, *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 8 shows that the rs1421085 T>C mutation leads to an increase in the expression level of FTO;
  • A-D FTO mRNA (left) and protein (right) expression in different point mutation models:
  • A) Point mutations and controls constructed by CRISPR/Cas9 Mice (n 6:7);
  • B) Point mutation and control mice constructed by ES method (n 3:3);
  • C) UKI and control mice (n 3:3);
  • D ES Methods were used to construct point mutation and control mouse BAT-derived SVF in vitro.
  • Figure 9 shows WB detection of thermogenesis-related gene expression levels (top); Seahorse detection of brown fat cell oxygen consumption (bottom).
  • Figure 10 is a structural diagram of KI identification.
  • Figure 11 shows the identification results of UCP1-Cre.
  • mice were raised by Shanghai Nanmo Biotechnology Co., Ltd.
  • the circadian rhythm of mice is maintained at 12-12 hours, with the lights on from 7:00am to 19:00pm and the lights off from 19:00pm to 7:00am.
  • the relative humidity of the environment is maintained at 55 ⁇ 10%, and the temperature is maintained at 21 ⁇ 1°C. .
  • Drinking water is disinfected by high temperature and pressure, feed is disinfected by ultraviolet irradiation for 12 hours, and water and food are fully supplied.
  • a normal diet was carried out using standard commercial mouse chow (4.5% fat, 4% fiber, 21% protein, 1.404 kcal/g) and high-fat chow purchased from Research Diet (60% fat, 20% carbohydrate, 20% protein).
  • mice After weaning (about 4 weeks old), the mice were divided into cages. 4-5 mice were fed to a cage with normal diet, and 3-4 mice were fed with high-fat diet to a cage. After the mice were divided into cages, their body weights were measured and recorded once a week.
  • the mouse FTO gene (NM_011936.2, Ensembl: ENSMUSG00000055932) is located on mouse chromosome 8, with a total length of 473084bp.
  • the obesity-related SNP rs1421085 (T>C) is located in the intron 1 region of the human FTO gene.
  • the mutation site is 64079bp away from the FTO promoter.
  • the donor gene sequence (GGCATCGCATTGATT, shown in SEQ ID NO.1) containing rs1421085 was constructed through homology and homology, and it was microinjected together with Cas9 mRNA and sgRNA (small guiding RNA) in vitro It was introduced into fertilized eggs and then injected into surrogate female mice to obtain KI (Knock-in) heterozygous (HE) mice (the process is shown in Figure 1). Mouse DNA was extracted and the PCR amplified products were sequenced to identify genotypes. An example of genotype sequencing results is shown in Figure 2.
  • the amplification primer sequence and sequencing primer sequence are as follows:
  • PCR primer mFTO-F CGAGCCCAGCAAACTCATTCCT (as shown in SEQ ID NO.2)
  • PCR primer mFTO-R CAGATTAAGGTGACGGGCTGGAT (as shown in SEQ ID NO.3)
  • Sequencing primer AAGGTGACATACACCAGGAGCC (as shown in SEQ ID NO.4)
  • CRISPR/Cas9 technology was first used to construct a C57BL/6J strain of mice with systemic homozygous rs1421085 T>C mutation (denoted as HO).
  • Wild-type mice (WT) were used as controls and were given normal diet and high-fat feeding respectively. There was no significant difference in body weight between the two groups of mice fed a normal diet. At 21 weeks of age, the average body weight of HO was 0.86g lighter than that of the control group, but there was no statistical difference (Figure 3A).
  • mice The same rs142085 T>C knock-in mice (HO mice) were constructed using ES targeting technology.
  • the body weight of adult male HO mice under normal diet feeding was not significantly different from that of the control group. However, after high-fat feeding, the body weight increased more slowly than that of the control group. There was a difference in the weight of the two groups at the 6th week of high-fat feeding. The average weight of the HO group was lower than that of the control group. The control was about 1.9g lighter. The weight difference between the two groups was about 3g in the 13th week of high-fat. The difference expanded to 3.6g in the 16th week of high-fat. The weight of the WT group was 46.7g (an increase of 82.2%), and that of the HO group. 43.1g (71.6% increase) as shown in Figure 4A.
  • thermogenesis-related genes in brown adipose tissue.
  • Prdm16, Dio2, Elovl6, etc. were all significantly increased (Figure 4H-J).
  • iBATx interscapular BAT removal by surgical operation (iBATx) was performed on point mutant mice: at 6 weeks of age, the mice were anesthetized and bilateral iBAT was removed and then sutured After the wound was stable for 2 weeks, high-fat feeding was started at the age of 8 weeks.
  • the primer sequences for KI genotype identification are as follows, the LoxP amplification product is ⁇ 357bp, and the KI amplification product is ⁇ 330b.
  • the primer sequence for UCP1-Cre (Jax-024670) genotype identification is as follows, the PCR product TG is about 113bp
  • the PCR reaction system is as follows:
  • Examples 1-3 verified that rs1421085 T>C plays an important role in high-fat-induced obesity. Therefore, the FTO rs1421085 T>C point mutation mouse model is used to study obesity-related diseases and is a useful tool for the development of related drugs. Preparation is facilitated.
  • mice carrying the rs1421085 T>C mutation were slightly but significantly increased compared with the control (cas9 was 1.59 times; ES was 1.50 times; UKI was 1.40 times; in vitro 1.12 times), and their relative protein expression levels were average Approximately 2 times that of the control group (1.56 times for cas9; 2.87 times for ES; 1.97 times for UKI; 1.82 times in vitro), all suggesting that rs1421085 can promote the increase in FTO transcription levels (Figure 8A-D). This indicates that rs1421085 promotes thermogenesis by increasing the expression level of FTO in brown fat, and ultimately resists high-fat-induced obesity.

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Abstract

一种FTO rs1421085 T>C 点突变小鼠模型及其应用，所述的FTO rs1421085 T>C 点突变小鼠模型为在筛选或制备抵抗肥胖药物提供了有效的实验动物模型，具有较好的应用前景。

Description

一种FTO rs1421085 T＞C点突变小鼠模型及其应用 技术领域
本发明属于动物模型领域，特别涉及一种FTO rs1421085 T>C点突变小鼠模型及其应用。
背景技术
随着经济的飞速发展与物质供给水平的提高，过剩的营养摄入与日趋减少的体力活动导致的能量稳态失衡成为人群整体性体重增加(体重分布曲线右移)的主要原因。近年来科技发展的进步也使我们对肥胖及代谢性疾病的了解更加深入，肥胖的危害性广为人知，人们对于饮食、运动和体重的重视程度日益提升，旨在减少卡路里摄入并增加能量消耗的生活方式和行为干预(如代谢性手术、轻断食等)虽有一定效果，但功效有限；与此同时，体内的各项激素参与的代谢调控系统及神经化学适应机制共同参与的机体防御机制导致了体重“易增难减”和减重后反弹等情况好发，更增加了肥胖治疗的难度。基于此，肥胖遗传学相关研究成为疾病早期防控与治疗的突破口。来自双胞胎和收养儿童相关的观察性研究发现，BMI的可遗传性约为40–70％。瘦素受体(leptinreceptor)、黑素皮质素受体(melanocortin receptor)等基因上突变位点与肥胖发生间关联(单基因性肥胖，monogenic obesity)的建立为肥胖遗传学研究带来了曙光。虽然仅从遗传学角度并无法完全解释近30年全球范围内肥胖人群的快速扩张，但或许可以解释在相同环境下个体间出现体重差异的原因，即，个体在整体人群体重曲线的相对位置主要是由遗传因素决定的，无论是在社会经济发展较低水平或较高水平；同时肥胖易感基因的发现可以加深我们对于机体体重调节和脂肪分布差异的病理生理机制的了解，为肥胖的治疗和预防提供新思路。虽然肥胖的发生是遗传、环境和社会心理等多因素共同作用的结果，但遗传学因素因其特有的不可调控性、外显率波动范围大且易受环境因素影响等问题，逐渐成为肥胖研究领域的难点。肥胖遗传学研究在肥胖发病、精准治疗、预防预判等方面的意义显得尤为重要。
发明内容
本发明所要解决的技术问题是提供一种FTO rs1421085 T>C点突变小鼠模型及其应用。
本发明的一种FTO rs1421085 T>C点突变小鼠模型在研究肥胖疾病中的应用。
所述应用包括但不限于药物筛选、诊断靶标发掘。
本发明的一种FTO rs1421085 T>C点突变小鼠模型在筛选或制备抵抗肥胖药物中的应用。
本发明的一种FTO rs1421085 T>C点突变小鼠模型在筛选或制备抵抗高脂诱导的肥胖药物中的应用。
所述小鼠模型为基于CRISPR/Cas的FTO rs1421085 T>C点突变小鼠模型、基于ES打 靶的FTO rs1421085 T>C点突变小鼠模型、棕色脂肪条件性FTO rs1421085 T>C点突变小鼠模型中的一种或几种。
所述基于CRISPR/Cas的FTO rs1421085 T>C点突变小鼠模型的构建方法为：构建包含rs1421085的供体基因序列，并在体外将其与Cas9 mRNA,sgRNA一起通过显微注射的方式导入受精卵内，随后注射至代孕雌性小鼠，鉴定基因型，得到FTO rs1421085 T>C点突变小鼠模型。
所述基于ES打靶的FTO rs1421085 T>C点突变小鼠模型的构建方法为：构建包含rs1421085的供体基因序列，在小鼠胚胎干细胞ES细胞中进行DNA同源重组后，将ES细胞重新注射到囊胚腔中形成嵌合胚胎，在假孕雌性小鼠体内发育，鉴定基因型，得到FTO rs1421085 T>C点突变小鼠模型。
所述Cre/Loxp系统构建棕色脂肪条件性FTO rs1421085 T>C点突变小鼠模型的构建方法：选择插入位点:利用UCSC在线数据库在SNP(rs1421085)上下游查找SNP附近(条件性突变区域≤3kb)的非保守区域。在该SNP上游2.6kb处查找到非保守区域，作为1st loxP的插入位点；下游0.3kb处查找到非保守区域，作为2ndloxP的插入位点。
在靶向载体中，1号内含子包含该SNP rs1421085的部分区域被反向克隆到2号外显子的上游；该区域侧翼分别为loxP和lox2272位点；C57BL/6品系小鼠的ES细胞将作为靶向载体插入的目标细胞。
有益效果
本发明首次提供了FTO rs1421085 T>C点突变小鼠模型及构建方法。并且本发明构建的FTO rs1421085 T>C点突变小鼠模型为在筛选或制备抵抗肥胖药物提供了有效的实验动物模型，具有较好的应用前景。
本发明三种FTO rs1421085 T>C点突变小鼠模型联合构建对肥胖发病、精准治疗、预防预判等方面具有重要意义。
附图说明
图1为CRISPR/Cas技术构建FTO rs1421085 T>C点突变小鼠原理示意图；
图2为野生型小鼠(上)与HE小鼠(下)DNA测序结果；
图3为与对照相比，雄性rs1421085突变小鼠代谢改善，能更好地抵抗HFD诱导的肥胖；(A)NCD组HO与对照组小鼠体重曲线图(左)与体重增长曲线图(右)。6周龄开始称重；n＝10:15。(B)HFD组HO与对照组小鼠体重曲线图(左)与体重增长曲线图(右)。6周龄开始称重，8周龄HFD喂养；n＝11:16。(C-D)HFD喂养第15周时HO与对照组小鼠葡萄 糖耐量实验(C)与曲线下面积(D)；n＝17。(E-F)HFD喂养第17周时胰岛素耐量实验(E)与曲线下面积(F)；n＝17。(G-J)32周龄时HO与对照小鼠的血清TC(G)、TG(H)、LDL(I)、HDL(J)水平；n＝11:16。所有数据以均数±标准误(Mean±SEM)表示，采用双尾t检验计算统计学差异，*p<0.05，**p<0.01。
图4为ES雄性rs1421085 T>C全身突变小鼠较对照组棕色脂肪组织产热能力增强；(A)NCD喂养(左，n＝8:8)与HFD喂养(右,n＝13:10)下HO与对照组小鼠体重变化曲线图。(B)HFD喂养2月时HO与对照组小鼠的日均摄食量，n＝4:6。(C-E)高脂15周后HO与对照组体脂、瘦组织重量占体重的质量分数(C)，BAT质量分数(D)，iWAT、eWAT和肝脏的质量分数(E)；n＝13：10。(F)iBAT与iWAT的HE染色拍照，图中所示标尺为200um。(G)BAT电镜照片，图中标尺为1um，红色箭头指示线粒体。(H)q-PCR检测BAT中产热及炎症相关基因的mRNA表达水平；n＝12：13。(I)Western-blot检测BAT中Ppar-γ、Pgc1-α、Ucp1蛋白的表达水平；n＝3：3。(J)图(H)中Ucp1蛋白相对表达水平定量(以Hsp90蛋白表达水平进行标化)。所有数据以均数±标准误(Mean±SEM)表示，采用双尾t检验计算统计学差异，*p<0.05，**p<0.01，***p<0.001。
图5为rs1421085全身突变小鼠抵抗高脂依赖经典BAT；：(A)iBAT切除及HFD喂养时间轴。(B)iBAT切除术后小鼠24周龄处死时大体照片。(C)iBAT切除术后进行高脂喂养的HO与对照小鼠体重曲线图；n＝7：9。(D)iBAT切除术后进行高脂喂养的HO与对照小鼠体重增长曲线图，y轴表示对应周龄体重与8周龄体重的差值；n＝7：9。(E)iBAT切除术后7～9周小鼠日均摄食量；n＝7：9。(F-H)iBAT切除术后小鼠24周龄处死时体脂(J),瘦组织(K)及iWAT、eWAT和肝脏(L)占体重的百分比；n＝7：9。所有数据以均数±标准误(Mean±SEM)表示，采用双尾t检验计算统计学差异，*p<0.05，**p<0.01。
图6为UKI(Ucp1-Cre；rs1421085 Knock-in)小鼠构建；
图7为雄性UKI小鼠抵抗高脂诱导的肥胖；(A)UKI与对照组小鼠体重变化曲线图，4周龄开始称重，9周龄开始HFD喂养；n＝12:7。(B-D)高脂2月后HO与对照组体脂、瘦组织重量占体重的质量分数(B)，BAT质量分数(C)，iWAT、eWAT和肝脏的质量分数(D)；n＝12:7。(E)iBAT与iWAT的HE染色拍照，图中所示标尺为200um。(F)BAT电镜照片，图中标尺为1um，红色箭头指示线粒体。(G)q-PCR检测BAT中产热及分化相关基因的mRNA表达水平；n＝14：9。(H)左图为Western-blot检测BAT中Ucp1、Pgc1-α、Ppar-γ、C/ebpα蛋白的表达水平，右图为蛋白相对表达水平定量(以Hsp90蛋白表达水平进行标化)；n＝3：3。所有数据以均数±标准误(Mean±SEM)表示，采用双尾t检验计算统计学差异，*p<0.05，**p<0.01。
图8为rs1421085 T>C突变导致FTO表达水平升高；：(A-D)不同点突变模型中FTO mRNA(左)及蛋白(右)表达情况：(A)CRISPR/Cas9方式构建的点突变及对照小鼠(n＝6：7)；(B)ES方式构建点突变及对照小鼠(n＝3：3)；(C)UKI及对照小鼠(n＝3：3)；(D)ES方式构建点突变及对照小鼠BAT来源SVF体外诱。
图9为WB检测产热相关基因表达水平(上)；Seahorse检测棕脂细胞耗氧量(下)。
图10为KI鉴定结构图。
图11为UCP1-Cre鉴定结果图。
具体实施方式
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解，在阅读了本发明讲授的内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
实验小鼠由上海南模生物饲养。小鼠饲养昼夜节律为12—12小时，开灯时间7:00am-19:00pm,关灯时间19:00pm-7:00am,环境相对湿度保持在55±10％，温度维持在21±1℃。饮水高温高压消毒，饲料紫外辐照12小时消毒，水和食物充分供给。正常饮食使用标准商业小鼠饲料(4.5％脂肪，4％纤维素，21％蛋白质、1.404kcal/g)高脂饲料购自Research Diet(60％脂肪，20％碳水化合物，20％蛋白质)。小鼠断奶后(约4周龄)分笼，正常饮食喂养小鼠4-5只一笼，高脂饮食喂养小鼠3-4只一笼。小鼠分笼后每周测量并记录一次体重。
实施例1
(1)CRISPR/Cas技术构建FTO rs1421085 T>C点突变小鼠模型
构建原理及策略:小鼠FTO基因(NM_011936.2,Ensembl:ENSMUSG00000055932)位于小鼠8号染色体上，全场473084bp.肥胖相关SNP rs1421085(T>C)位于人类FTO基因1号内含子区域，该突变位点距离FTO启动子64079bp。
通过同源同组的方式构建包含rs1421085的供体基因序列(GGCATCGCATTGATT，如SEQ ID NO.1所示)，并在体外将其与Cas9 mRNA,sgRNA(small guiding RNA)一起通过显微注射的方式导入受精卵内，随后注射至代孕雌性小鼠，得到KI(Knock-in)杂合(HE)小鼠(过程示意见图1所示)。提取小鼠DNA并进行PCR扩增的产物进行测序，以鉴定基因型。基因型测序结果示例见图2所示。
扩增引物序列、测序引物序列如下：
PCR引物mFTO-F：CGAGCCCAGCAAACTCATTCCT(如SEQ ID NO.2所示)
PCR引物mFTO-R：CAGATTAAGGTGACGGGCTGGAT(如SEQ ID NO.3所示)
测序引物：AAGGTGACATACACCAGGAGCC(如SEQ ID NO.4所示)
先采用CRISPR/Cas9技术构建了全身性纯合rs1421085 T>C突变的C57BL/6J品系小鼠(记为HO)，以野生型小鼠(WT)作为对照，分别给予正常饮食及高脂喂养。正常饮食喂养下两组小鼠未见明显体重差异，21周龄时HO平均体重比对照组轻0.86g,但并无统计学差异(图3A)。而在高脂喂养3周后，两组小鼠开始出现体重增长差异，且有随时间增长而逐渐扩大趋势：在高脂喂养5周龄时HO组平均体重较对照组轻约3.6g，21周龄时两组间体重差异已扩大至5.68g(图3B)。高脂喂养的3月内，WT组平均体重共增加约23.6g(84.4％),而HO组平均体重增加约18.7克(80.7％)。高脂喂养下HO组体重增加趋缓提示机体代谢情况的改善，因此我们检测了HFD喂养后小鼠的糖脂代谢相关指标。结果发现，HO小鼠糖代谢较对照组改善，基因型间葡萄糖耐量及胰岛素耐量的差异均以空腹、注射后30分钟和60分钟检测时间点较为显著；同时HO组血脂代谢水平较对照组亦有改善，其总胆固醇(TC)水平、低密度胆固醇(LDL)水平均显著低于对照组(图3C-J)。
实施例2
ES打靶构建rs1421085突变小鼠：
采用ES打靶技术构建了同样的rs142085 T>C敲入小鼠(HO小鼠)。
成年雄性HO小鼠在正常饮食喂养下体重与对照无显著差异，而在高脂喂养后体重上升较对照组更为缓慢，高脂喂养第6周时两组体重出现差异，HO组平均体重较对照轻约1.9g，在高脂第13周时两组的体重差异约为3g，高脂第16周时该差异扩大至3.6g，WT组体重为46.7g(增长82.2％)，HO组为43.1g(增长71.6％)见图4A。在两组小鼠高脂喂养2个月时进行了摄食量检测，发现两组间并未出现显著差异(图4B)。小鼠达25周龄时(高脂16周)检测其体脂率，HO组体脂率显著低于对照，而瘦组织百分比略高于对照(图4C)。组织称重结果显示，HO组小鼠肝脏质量分数显著低于对照，而棕色脂肪组织和皮下白色脂肪组织重量较对照组有降低趋势但并未出现统计学差异(图4D-E)。HE染色切片及电镜结果可见，HO组小鼠棕色脂肪细胞内出现了数量更多的细小的脂滴及更多的线粒体，而皮下脂肪内亦有脂滴缩小的改变(图4F-G)。进一步用qPCR和Western-blot分析其棕色脂肪组织内产热相关基因的mRNA及蛋白表达水平，HO组Ucp1，Pgc1-α，Cebp/α，Ppar-γ及其他参与产热及线粒体功能相关的基因如Prdm16，Dio2,Elovl6等均显著升高(图4H-J)。
为阐明棕色脂肪在rs1421085点突变抵抗肥胖中的作用，对点突变小鼠进行了iBATx(通过外科手术(iBATx)切除肩胛间BAT)：在6周龄时麻醉并切除小鼠双侧iBAT后缝合伤口，稳定2周后于8周龄对其开始高脂喂养。术后HO组的体重较对照组不再减轻，反而出现了升高的情况；两组间体重开始出现差异的时间较非手术组较晚(高脂9周时显现)，且组间差异较小，在22周龄时(高脂13周)两组体重相差约5.5g(图5C-D)。两组小鼠的摄食 量仍无显著差异。HO组体重的增加主要由体脂(尤其是iWAT)贡献。HE染色分析显示HO组iWAT脂滴面积更大，而eWAT与对照无显著差异(图5)。IBATx小鼠表型充分说明，全身点突变小鼠产热功能增加、抵抗高脂诱发肥胖依赖或部分依赖于经典棕色脂肪组织的存在。
实施例3
两种全身性rs1421085 T>C突变小鼠以及BAT缺失模型的表型特征提示，rs1421085突变调节体重的关键效应组织为BAT。为了明确棕色脂肪组织是否为该SNP的直接效应器官，利用Cre/loxp系统构建了第三个点突变模型，即棕色脂肪组织特异性rs1421085 T>C突变小鼠模型(Ucp1-cre；FTO rs1421085 T＞CKnock-in,UKI)。构建策略和模式图详见方法部分(如图6所示)。
KI基因型鉴定引物序列如下，LoxP扩增产物～357bp,KI扩增产物～330b。
PCR组分:
PCR程序如下表所示：
KI鉴定结果如图10所示。
UCP1-Cre(Jax-024670)基因型鉴定引物序列如下，PCR产物TG约113bp
引物  5’到3’碱基序列
P1    GTT CTT GCA CTC ACG CCT CT(如SEQ ID NO.9所示)
P2    TGC GAA CCT CAT CAC TCG T(如SEQ ID NO.10所示)；
PCR反应体系如下表：
循环数及反应温度：
UCP1-Cre鉴定结果如图11所示。
雄性UKI与对照小鼠(KIfl/fl)成年后进行高脂喂养,UKI组体重增加少于对照组，两组间体重差异随高脂时间增长而扩大，在高脂8周时两组间平均体重相差约4.05g，对照组平均体重为41.9g(增长60.1％)，HO组平均体重为31.8g(增长49.8％)，如图7A。UKI小鼠体脂显著低于对照，但两组的组织称重并无显著差异(图7B-D)。棕色脂肪的HE染色及电镜切片结果均可观察到与全身性点突变小鼠类似的脂滴缩小，线粒体数目增多等形态学改变(图7E-F)。UKI组棕色脂肪组织的Ucp1 mRNA表达水平显著升高，约为对照的1.7倍，其蛋白表达水平也出现了增高趋势；同时，C/ebpα蛋白表达水平显著升高，Pgc1-α蛋白也 出现了增高趋势(图7G-H)。
实施例1-3验证了rs1421085 T>C在高脂诱导的肥胖疾病中起着重要的作用，因此FTO rs1421085 T>C点突变小鼠模型用于研究肥胖相关疾病的模型，并为相关药物的制备提供了便利。
实施例4
携带rs1421085 T>C突变的小鼠FTO mRNA表达水平均较对照有微弱但较为显著的增加(cas9为1.59倍；ES为1.50倍；UKI为1.40倍；体外1.12倍)，其蛋白相对表达量平均约为对照组的2倍(cas9为1.56倍；ES为2.87倍；UKI为1.97倍；体外1.82倍)，均提示rs1421085可促进FTO转录水平增加(图8A-D)。说明rs1421085通过增加棕脂中FTO表达水平而促进产热，最终抵抗高脂诱导的肥胖。
分离了野生型和ES点突变小鼠棕脂来源的SVF，在用慢病毒敲低FTO的基础上，进行诱导分化，如图9所示可知，rs1421085 T>C突变造成的棕色脂肪细胞产热能力增强的表型部分由FTO蛋白介导。

Claims (7)

  1. 一种FTO rs1421085 T>C点突变小鼠模型在研究肥胖疾病中的应用。
  2. 一种FTO rs1421085 T>C点突变小鼠模型在筛选或制备抵抗肥胖药物中的应用。
  3. 根据权利要求2所述应用，其特征在于，所述FTO rs1421085 T>C点突变小鼠模型在筛选或制备抵抗高脂诱导的肥胖药物中的应用。
  4. 根据权利要求1-2任一所述的应用，其特征在于，所述小鼠模型为基于CRISPR/Cas的FTO rs1421085 T>C点突变小鼠模型、基于ES打靶的FTO rs1421085 T>C点突变小鼠模型、棕色脂肪条件性FTO rs1421085 T>C点突变小鼠模型中的一种或几种。
  5. 根据权利要求1所述小鼠模型，其特征在于，所述基于CRISPR/Cas的FTO rs1421085 T>C点突变小鼠模型的构建方法为：构建包含rs1421085的供体基因序列，并在体外将其与Cas9 mRNA,sgRNA一起通过显微注射的方式导入受精卵内，随后注射至代孕雌性小鼠，鉴定基因型，得到FTO rs1421085 T>C点突变小鼠模型。
  6. 根据权利要求1所述小鼠模型，其特征在于，所述基于ES打靶的FTO rs1421085 T>C点突变小鼠模型的构建方法为：构建包含rs1421085的供体基因序列，在小鼠胚胎干细胞ES细胞中进行DNA同源重组后，将ES细胞重新注射到囊胚腔中形成嵌合胚胎，在假孕雌性小鼠体内发育，鉴定基因型，得到FTO rs1421085 T>C点突变小鼠模型。
  7. 根据权利要求1所述小鼠模型，其特征在于，所述棕色脂肪条件性FTO rs1421085 T>C点突变小鼠模型的构建方法：选择插入位点；在靶向载体中，1号内含子包含该SNP rs1421085的部分区域被反向克隆到2号外显子的上游；该区域侧翼分别为loxP和lox2272位点；C57BL/6品系小鼠的ES细胞将作为靶向载体插入的目标细胞。
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