CN106148370A - 肥胖症大鼠动物模型和构建方法 - Google Patents

肥胖症大鼠动物模型和构建方法 Download PDF

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CN106148370A
CN106148370A CN201610445838.0A CN201610445838A CN106148370A CN 106148370 A CN106148370 A CN 106148370A CN 201610445838 A CN201610445838 A CN 201610445838A CN 106148370 A CN106148370 A CN 106148370A
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祝献民
范国平
曾维鸣
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SUZHOU RUIQI BIOLOGICAL MEDICINE SCIENCE & TECHNOLOGY Co Ltd
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Abstract

本发明涉及肥胖症大鼠动物模型和构建方法，属于动物基因工程和遗传修饰领域，主要通过CRISPR/Cas9技术获得LEP和LEPR基因敲除的大鼠，LEP和LEPR基因敲除大鼠具有典型肥胖特征，可为人类肥胖疾病研究提供一种可靠的动物模型，有望成为肥胖大鼠的一种标准化实验动物。

Description

肥胖症大鼠动物模型和构建方法
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，涉及一种基于基因敲除技术建立的肥胖症疾病大鼠动物模型和构建方法。
背景技术
随着生活水平的提高，肥胖症(obesity)作为一种慢性病，发病率急剧升高，成为世界性的医疗难题。肥胖症还与糖尿病关系密切，40岁以上的糖尿病患者中有70％～80％在患病前已有肥胖症。同时，肥胖症还伴有高脂血症、高血压、糖耐量异常等症状，并成为动脉硬化的主要诱因。Leptin(Lep)是1994年发现的一种由脂肪细胞分泌的激素。通过中枢神经系统的Leptin受体(Lepr)，Lep可以调控包括血糖浓度、神经内分泌、体重、食欲等多种生理功能和个体行为。目前的研究表明，Lep对体重的调控主要是通过GABA能神经元起作用的；而Lep在下丘脑的不同部位起到不同的作用：青春期葡萄糖稳态的调控在腹侧前乳头核；与重度肥胖症及胰岛素抗性则发生在弓形核中的表达亲阿黑皮素原(POMC)的神经元中。由于Lep及Lepr在糖、脂代谢中的重要作用，已经有多种针对它们及其介导途径的临床药物研究。其中小鼠模型是使用最为广泛的模型，包括针对Lep的ob/ob小鼠模型和针对Lepr的db/db小鼠模型。与小鼠相比，大鼠是研究营养学的常用模式生物。然而，由于种种原因，目前为止只有对应于db/db 小鼠(Lepr缺陷)的大鼠模型，而尚无对应ob/ob小鼠(Lep缺陷)的大鼠模型。不仅如此，以前建立的模型不仅有定点突变，还包括自发突变等随机方法，因此不利于系统阐述基因突变导致的功能缺失。还需要指出的是，以往的建模方法也很难建立多种基因同时突变的模型来研究基因之间的相互关系。
基因修饰动物(包括人源化动物)是研究生物医学领域中的分子机理和致病机制的重要研究手段。传统的转基因动物制作方法需要得到相应动物的干细胞，显微注射改造的遗传物质后将干细胞植入囊胚，直至生产出嵌合型动物，并通过杂交最终得到纯合型动物。整个过程不但耗时，而且由于缺乏各种动物的干细胞系，转基因动物的工作主要在小鼠中开展。虽然转基因小鼠的研究对于研究人类疾病有着重要作用，却不能完全模拟人类疾病的真实情况。新的CRISPR技术为制作转基因技术提供了一个崭新的平台。在最新报道中，Wang et al利用CRISPR/Cas系统直接改造受精卵的遗传物质，并通过辅助生殖技术得到靶向基因修饰动物。这项工作使整个工作缩短到一个月，而且可以同时对两个或多个基因同时进行修饰。
基于上述依据，本项专利用CRISPR/Cas系统同时敲除Lep两个等位基因，得到相应的靶向基因修饰的大鼠模型。本研究不仅可以加深对肥胖症发病过程中基因调控的认识，而且可以为转化医学和新药研发提供高水平的动物模型。
发明内容
本发明的目的是提供一种肥胖症大鼠动物模型，是利用
CRISPR/Cas9技术获得LEP/LEPR特异型基因敲除的大鼠。
本发明首先提供了大鼠LEP基因第2外显子和LEPR基因第2和第3外显子在制备肥胖症大鼠动物模型中的用途。所述大鼠LEP基因第2外显子的核苷酸序列如SEQ ID NO.5：5’-GTCCAAGAAGAAGAAGAAGA CCCCAGCGAG GAAAATGTGC TGGAGACCCC TGTGCCGGTTCCTGTGGCTTTGGTCCTATC TGTCCTATGTTCAAGCTGTG CCTATCCACA AAGTCCAGGATGACACCAAAACCCTCATCAAGACCATTGT CACCAGGATC AATGACATTTCACACACG-3’所示，所述大鼠LEPR基因第2外显子的核苷酸序列SEQ ID NO.6：5’-GTGTCTATCT CTGAAGTAAG ATGACGTGTC AGAAATTCTA TGTGGTTTTGTTACACTGGG-3’所示；所述大鼠LEPR基因第3外显子的核苷酸序列为序列表中SEQ ID NO.7所示。
本发明提供了大鼠LEP基因第2外显子和大鼠LEPR基因第2外显子和第3外显子的sgRNA在敲除大鼠LEP基因中的应用。
本发明还提供了含有针对上述sgRNA的DNA序列的载体。在本发明的实施例中，提供的上述载体为pX330。
特异性靶向LEP基因第2外显子或者LEPR基因第2或3外显子的sgRNA的表达载体，由载体pX330和与载体pX330连接的靶向DNA构成，靶向DNA序列为Seq ID No1-ss和Seq IDNo1-as、Seq ID No2-ss和Seq ID No2-as、Seq ID No3-ss和Seq ID No3-as、Seq ID No4-ss和Seq ID No4-as中的一对序列。
所述Seq ID No1-ss和Seq ID No1-as、Seq ID No2-ss和Seq ID No2-as分别针对LEP基因第2外显子的gRNA靶点，Seq ID No3-ss和Seq ID No3-as针对LEPR基因第2外显子的gRNA靶点，Seq ID No4-ss和Seq ID No4-as针对LEPR基因第3外显子的gRNA靶点。
本发明提供了用于敲除大鼠LEP或LEPR基因的CRISPR-Cas9表达系统，含有特异性靶向LEP基因第2外显子或LEPR基因第2或第3外显子的sgRNA的表达载体，pX330载体能表达Cas9 mRNA。
本发明还提供肥胖症大鼠动物模型的构建方法，包括以下步骤：
(1)构建特异性靶向LEP基因第2外显子或者LEPR基因第2或3外显子的sgRNA的表达载体；
(2)通过体外转录分别表达得到LEP基因第2外显子或者LEPR基因第2或3外显子的sgRNA，以及Cas9 mRNA；
(3)将sgRNA和Cas9 mRNA纯化后，混合，注射入大鼠受精卵细胞中，移植入大鼠输卵管中，以生产敲除LEP或LEPR基因的动物。
步骤(2)转录产生的Cas9 mRNA和sgRNA分别进行吸附柱纯化，将纯化后的Cas9mRNA和sgRNA分别利用分光光度计测定浓度。
步骤(3)中，sgRNA与Cas9 mRNA混合时，二者的质量比为1:2。
本发明提供了一种肥胖症大鼠动物模型，通过以下检测指标验证：
(1)Lep/Lepr基因敲除大鼠的进食量和饮水量增加、体重较大；
(2)血清中甘油三酯、胆固醇、高密度脂蛋白、低密度脂蛋白、胰岛素和葡萄糖水平升高；
(3)肝脏、肾脏和睾丸等组织异常。
本发明还提供一种非人类哺乳动物细胞，细胞内含有构建方法步 骤(2)得到的sgRNA和Cas9 mRNA。
本发明涉及肥胖症大鼠动物模型和构建方法，主要通过CRISPR/Cas9技术获得LEP和LEPR基因敲除的大鼠，LEP和LEPR基因敲除大鼠具有典型肥胖特征，为人类肥胖疾病研究提供一种可靠的动物模型，有望成为肥胖大鼠的一种标准化实验动物。
附图说明
图1为利用CRISPR/Cas9技术建立Lep基因突变大鼠的示意图；
图2图示了Lep突变引起的肥胖、糖耐受和高胰岛素血症；
图3为分别对Lep野生型大鼠和Lep突变大鼠的肝脏、肾脏和睾丸等组织进行HE染色，结果显示突变大鼠患有脂肪肝、肾病和不孕等症状。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，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，所用原料均为市售商品。
以下实施例中未作具体说明的分子生物学实验方法，均参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中描述的具体方法进行，或者按照试剂盒和产品说明书进行。
实施例1针对Lep的CRISPR-Cas9表达系统的构建(图1)
1、根据大鼠Lep和LEPR(leptin receptor)序列，进行sgRNA的设计并获得了sgRNA的序列信息。其中特异性靶向Lep基因第2外显子的sgRNA，其DNA序列如SEQ ID NO.1、SEQID NO.2所示；其 中特异性靶向Lepr基因第2和第3外显子的sgRNA，其DNA序列分别如SEQID NO.3和SEQ ID NO.4所示。其中SEQ ID NO.1、SEQ ID NO.2靶向SEQ ID NO.5(SEQ IDNO.5为LEP基因的一部分)，而SEQ ID NO.3、SEQ ID NO.4分别靶向SEQ ID NO.6和7(SEQ IDNO.6和7分别为LEPR基因的一部分)。SEQ ID NO.1-4每一个序列均为退火形成的双链DNA，以SEQ ID NO.1举例：分别合成正反单链Seq ID No1-ss和Seq ID No1-as，然后退火形成SEQ ID NO.1。
2、sgRNA载体质粒的构建：(1)设计并合成识别Lep基因第2外显子和LEPR基因第2和3外显子的sgRNA识别区DNA序列，如Seq ID No1-ss和Seq ID No1-as所示(同样地，SeqID No2-ss和Seq ID No2-as、Seq ID No3-ss和Seq ID No3-as、Seq ID No4-ss和Seq IDNo4-as分别为一对序列)，具体如表2，每个sgRNA载体质粒构建时，仅仅在载体中插入SEQID NO.1-4中的1对序列；(2)每个DNA先合成单链，合成后的单链sgRNA序列进行磷酸化后梯度降温退火，形成双链DNA；具体步骤为将合成的oligoDNA(以SEQ ID NO.1为例，SEQ IDNO1-ss作为正链oligoDNA，Seq ID No1-as作为反链序列，将正、反链oligoDNA与10×T4Ligation Buffer和T4PNK以2:2:1的体积比例混合后再加入3倍体积的水补齐体系，然后37℃孵育30min，再95℃，5min变性，之后在以5℃每分钟的速率降温至25℃完成反应以产生磷酸化粘性末端；同时BbsI酶切载体pX330产生粘性末端；退火反应体系如表1所示。
表1退火反应体系
表2sgRNA识别区DNA序列，其中，ss是正链，as是负链。
(3)以T4连接酶分别将SEQ ID NO.1-4的双链DNA分别与pX330进行连接，获得多个真核CRISPR-Cas9表达系统载体。连接反应体系如表3。连接反应的温度为16摄氏度，连接反应时间为1h-3h。
表3连接反应体系
实施例2体外转录
利用T7-Cas9PCR和T7-sgRNA PCR产物进行以T7启动子介导的体外转录，即以T7启动子作为体外转录的启动子，利用RNA聚合酶在体外实现从DNA到mRNA的转录过程，具体方法为：用mMESSAGE mMACHINE T7ULTRA kit(Life Technologies)体外转录 T7-Cas9PCR产物，用MEGAshortscript T7kit(Life Technologies)体外转录T7-sgRNA PCR产物。将转录产生的mRNA进行纯化，具体方法为：Cas9mRNA用MEGAclear kit(Life Technologies)纯化，sgRNAs用乙醇沉淀方法纯化，用纯水溶解mRNA，将纯化后的mRNA利用分光光度计测定浓度。T7-Cas9 PCR引物见表4，T7-sgRNA PCR引物见表5，gRNA-Lep1-F、gRNA-Lep2-F、gRNA-LepR1-F、gRNA-LepR2-F分别为针对SEQ ID NO.1-4的正向引物，反向引物均为gRNA-R。PCR反应体系见表6，PCR反应条件见表7。
表4T7-Cas9 PCR引物
表5T7-sgRNA PCR引物
表6PCR反应体系
表7PCR反应条件
实施例3利用针对Lep/LEPR的CRISPR-Cas9系统mRNA生产基因打靶大鼠
1、原核注射及胚胎移植
取大鼠的原核期受精卵，利用显微注射仪将预混好的Cas9mRNA/sgRNA混合物(Cas9mRNA终浓度为100ng/μl，sgRNA终浓度为50ng/μl)，注射至大鼠受精卵细胞质，然后移植至受体母鼠的输卵管中，生产基因打靶大鼠，注射量为0.5-1ul。
2、基因打靶大鼠的鉴定
代孕母鼠生产后，待仔鼠长至2周龄剪取1cm左右鼠尾，蛋白酶K，55℃消化后酚仿抽提提取鼠尾基因组。以鼠尾基因组为模板，分别设计针对Lep基因第2外显子和LEPR基因第2和3外显子的引物，进行扩增，对获得的PCR产物进行测序，引物序列如下表8。其中，LepF和Lep R对应于SEQ ID NO.5，Lepr1F和Lepr 1R对应于SEQ ID NO.6， Lepr2F和Lepr2R对应于SEQ ID NO.7。
表8基因打靶大鼠的鉴定引物
如测序结果打靶位点附近出现双峰的情况，则可能为打靶成功。选择双峰的样品再次PCR，产物胶回收后TA克隆至T载体中，转化后挑取阳性克隆再次进行测序，如测序结果Lep/LEPR基因靶位点附近发生碱基插入或碱基缺失，导致阅读框移码突变，则可判断为Lep/LEPR基因敲除。
图1C中，LEP1针对SEQ ID NO.1，LEP2针对SEQ ID NO.2，Lepr1针对SEQ ID NO.3，可知经过SEQ ID NO.1-3改造的基因打靶大鼠均发生了碱基缺失和/或插入。
Lep1
36,39,41,43,44
AGGATGACACCAAAACCCTC---AAGACCATTGTCACC(-3bp)
Lep2
17-2CA---AGACCATTGTCACC-------ATGACATTTCACACA(-3/-7bp)
17-2CAT(T)CAAGACCATTGTCA---------ATGACATTTCACACA(+1/-9bp)
Lepr1
23GACGTGTCAGAAATTC(T)TATGTGGTTTTGTTACACTGGG(+1bp)
25-1GACGTGTCAGAAATTCT--GTGGTTTTGTTACACTGGG(-2bp)
25-2GACGTGTCAGAAATTC(T)TATGTGGTTTTGTTACACTGGG(+1bp)
实施例4利用肥胖症检测指标验证一种肥胖症大鼠动物模型(图2和图3)：其中图2和图3均针对SEQ ID NO.1-4对应的大鼠模型；
(1)Lep基因敲除大鼠的进食量和饮水量增加、体重较大；
(2)Lep基因敲除大鼠血清中甘油三酯、胆固醇、高密度脂蛋白、低密度脂蛋白、胰岛素和葡萄糖水平升高，如表9所示；
(3)Lep基因敲除大鼠肝脏、肾脏和睾丸等组织异常。
表1 Lep基因敲除大鼠血清中甘油三酯、胆固醇、高密度脂蛋白、低密度脂蛋白、胰岛素和葡萄糖水平
表9 Lep基因敲除大鼠血清中甘油三酯、胆固醇、高密度脂蛋白、低密度脂蛋白、胰岛素和葡萄糖水平
所有数值为means±SD.(雄性大鼠:野生型n＝4；Lep突变n＝7。雌性大鼠:野生型n＝3；Lep突变n＝7)
*与野生型对比，p<0.05.
虽然，上文中已经用一般性说明、具体实施方式及试验，对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (8)

1. 特异性靶向LEP基因第2外显子或者LEPR基因第2或3外显子的sgRNA的表达载体，其特征在于：由载体pX330和与载体pX330连接的靶向DNA构成，靶向DNA序列为Seq ID No1-ss和Seq ID No1-as、Seq ID No2-ss和Seq ID No2-as、Seq ID No3-ss和Seq ID No3-as、SeqID No4-ss和Seq ID No4-as中的一对序列。
2. 根据权利要求1所述的特异性靶向LEP基因第2外显子或者LEPR基因第2或3外显子的sgRNA的表达载体，其特征在于：所述 Seq ID No1-ss和Seq ID No1-as、Seq ID No2-ss和Seq ID No2-as分别针对LEP基因第2外显子的gRNA 靶点，Seq ID No3-ss和Seq ID No3-as针对LEPR基因第2外显子的gRNA靶点， Seq ID No4-ss和Seq ID No4-as针对LEPR基因第3外显子的gRNA靶点。
3.肥胖症大鼠动物模型的构建方法，包括以下步骤：
(1) 构建权利要求1或2所述的特异性靶向LEP基因第2外显子或者LEPR基因第2或3外显子的sgRNA的表达载体；
(2) 通过体外转录分别表达得到LEP基因第2外显子或者LEPR基因第2或3外显子的sgRNA，以及Cas9 mRNA；
(3) 将sgRNA和Cas9 mRNA纯化后，混合，注射入大鼠受精卵细胞中，移植入大鼠输卵管中，以生产敲除LEP或LEPR基因的动物。
4. 根据权利要求3所述的肥胖症大鼠动物模型的构建方法，其特征在于，步骤(2)转录产生的Cas9 mRNA和sgRNA分别进行吸附柱纯化，将纯化后的Cas9 mRNA和sgRNA分别利用分光光度计测定浓度。
5. 根据权利要求3所述的肥胖症大鼠动物模型的构建方法，其特征在于，步骤(3)中，sgRNA与Cas9 mRNA混合时，二者的质量比为1:2。
6. 特异性靶向LEP基因第2外显子或者LEPR基因第2或3外显子的sgRNA的靶向DNA序列，其特征在于，靶向DNA序列为Seq ID No1-ss和Seq ID No1-as、Seq ID No2-ss和Seq IDNo2-as、Seq ID No3-ss和Seq ID No3-as、Seq ID No4-ss和Seq ID No4-as中的一对序列。
7.权利要求3所述的构建方法在Lep/Lepr基因敲除大鼠模型中的应用。
8. 一种非人类哺乳动物细胞，细胞内含有权利要求3所述的sgRNA和Cas9 mRNA。
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