CN103614415A - 一种基于crispr基因敲除技术建立肥胖症大鼠动物模型的方法 - Google Patents
一种基于crispr基因敲除技术建立肥胖症大鼠动物模型的方法 Download PDFInfo
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Abstract
一种基于CRISPR基因敲除技术建立肥胖症大鼠动物模型的方法,包括以下步骤:(1)建立Lep/Lepr基因敲除大鼠模型;(2)肥胖症疾病大鼠动物模型的鉴定及相关分析;(3)肥胖症疾病大鼠动物模型的能量代谢与体脂率评价。本发明利用CRISPR/Cas系统分别或同时敲除Lep和LepR两个基因,得到相应的靶向基因修饰的大鼠模型,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。
Description
技术领域
本发明涉及生物医学领域,具体为一种基于CRISPR基因敲除技术建立肥胖症大鼠动物模型的方法。
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,并成为动脉粥样硬化的主要诱因。瘦素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,转基因动物的工作目前主要在小鼠中开展。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。
发明内容
本发明针对现有肥胖症疾病动物模型的不足,公开了一种用CRISPR基因敲除技术建立肥胖症动物模型的方法,以期阐明肥胖症发病机制。
为实现上述目的,本发明提供如下技术方案:
一种基于CRISPR基因敲除技术建立肥胖症大鼠动物模型的方法,包括以下步骤:
(1)建立Lep/Lepr基因敲除大鼠模型;
(2)肥胖症疾病大鼠动物模型的鉴定及相关表型分析;
(3)肥胖症疾病大鼠动物模型的能量代谢与体脂率评价。
所述建立Lep/Lepr基因敲除大鼠模型,具体包括以下步骤:
1)CRISPR靶向修饰基因载体的构建;
2)大鼠促排卵和体外受精;
3)大鼠受精卵的显微注射;
4)受精卵体外培养、植入受体及靶向基因修饰动物的培育。
所述肥胖症疾病大鼠动物模型的鉴定及相关分析,具体包括以下步骤:
1)对CRISPR靶向位点进行DNA测序鉴定大鼠动物模型的基因型;
2)利用实时定量PCR(real-time PCR)检测大鼠细胞中Lep/Lepr基因mRNA水平的变化;
3)通过免疫荧光染色检测大鼠细胞中Lep/Lepr蛋白的缺失;
4)观察大鼠心脏、肝、脾脏、肾脏、肌肉等重要器官或组织是否发生病变;
5)观察大鼠不同部位脂肪量和肝脏重量是否发生变化。
所述肥胖症疾病大鼠动物模型的能量代谢与体脂率评价,具体包括以下步骤:
1)实验期间定期观察大鼠进食量、撒食量、身长、尾长、体重,并计算食物利用率和Lees指数;
2)采用体脂率直接测量法测量体脂率:利用人体组成分析仪和生物电阻抗技术﹙BIA﹚法测定体脂率;
3)采用体脂率间接测量法测量体脂率:利用皮褶厚度计测量受试对象的臂部、肩胛部、腹部、髂部、大腿部5个部位皮褶厚度。
所述食物利用率计算公式为:食物利用率=体重增长量/(喂食量-撒食量)。
所述和Lees指数计算公式为:Lees指数=体重(g)×103/体长(cm) 。
本发明与以往技术相比,具有以下优点:
本发明利用CRISPR/Cas系统分别或同时敲除Lep和LepR两个基因,得到相应的靶向基因修饰的大鼠模型,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。
具体实施方式
下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
一种基于CRISPR基因敲除技术建立肥胖症大鼠动物模型的方法,包括以下步骤:
(1)建立Lep/Lepr基因敲除大鼠模型;
(2)肥胖症疾病大鼠动物模型的鉴定及相关分析;
(3)肥胖症疾病大鼠动物模型的能量代谢与体脂率评价。
所述建立Lep/Lepr基因敲除大鼠模型,具体包括以下步骤:
1)CRISPR靶向修饰基因载体的构建;
2)大鼠促排卵和体外受精;
3)大鼠受精卵的显微注射;
4)受精卵体外培养、植入受体及靶向基因修饰动物的培育。
所述肥胖症疾病大鼠动物模型的鉴定及相关分析,具体包括以下步骤:
1)对CRISPR靶向位点进行DNA测序鉴定大鼠动物模型的基因型;
2)利用实时定量PCR(real-time PCR)检测大鼠细胞中Lep/Lepr基因mRNA水平的变化;
3)通过免疫荧光染色检测大鼠细胞中Lep/Lepr蛋白的缺失;
4)观察大鼠心脏、肝、脾脏、肾脏、肌肉等重要器官或组织是否发生病变;
5)观察大鼠不同部位脂肪量和肝脏重量是否发生变化。
所述肥胖症疾病大鼠动物模型的能量代谢与体脂率评价,具体包括以下步骤:
1)实验期间定期观察大鼠进食量、撒食量、身长、尾长、体重,并计算食物利用率和Lees指数;
2)采用体脂率直接测量法测量体脂率:利用人体组成分析仪和生物电阻抗技术﹙BIA﹚法测定体脂率;
3)采用体脂率间接测量法测量体脂率:利用皮褶厚度计测量受试对象的臂部、肩胛部、腹部、髂部、大腿部5个部位皮褶厚度。
所述食物利用率计算公式为:食物利用率=体重增长量/(喂食量-撒食量)。
所述和Lees指数计算公式为:Lees指数=体重(g)×103/体长(cm) 。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。
此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其他实施方式。
Claims (6)
1.一种基于CRISPR基因敲除技术建立肥胖症大鼠动物模型的方法,其特征是,包括以下步骤:
(1)建立Lep/Lepr基因敲除大鼠模型;
(2)肥胖症疾病大鼠动物模型的鉴定及相关分析;
(3)肥胖症疾病大鼠动物模型的能量代谢与体脂率评价。
2.根据权利要求1所述的一种基于CRISPR基因敲除技术建立肥胖症大鼠动物模型的方法,其特征是,所述建立Lep/Lepr基因敲除大鼠模型,具体包括以下步骤:
1)CRISPR靶向修饰基因载体的构建;
2)大鼠促排卵和体外受精;
3)大鼠受精卵的显微注射;
4)受精卵体外培养、植入受体及靶向基因修饰动物的培育。
3.根据权利要求1所述的一种基于CRISPR基因敲除技术建立肥胖症大鼠动物模型的方法,其特征是,所述肥胖症疾病大鼠动物模型的鉴定及相关分析,具体包括以下步骤:
1)对CRISPR靶向位点进行DNA测序鉴定大鼠动物模型的基因型;
2)利用实时定量PCR(real-time PCR)检测大鼠细胞中Lep/Lepr基因mRNA水平的变化;
3)通过免疫荧光染色检测大鼠细胞中Lep/Lepr蛋白的缺失;
4)观察大鼠心脏、肝、脾脏、肾脏、肌肉、视网膜等重要器官或组织是否发生病变;
5)观察大鼠不同部位脂肪量和肝脏重量是否发生变化。
4.根据权利要求1所述的一种基于CRISPR基因敲除技术建立肥胖症大鼠动物模型的方法,其特征是,所述肥胖症疾病大鼠动物模型的能量代谢与体脂率评价,具体包括以下步骤:
1)实验期间定期观察大鼠进食量、撒食量、身长、尾长、体重,并计算食物利用率和Lees指数;
2)采用体脂率直接测量法测量体脂率:利用人体组成分析仪和生物电阻抗技术﹙BIA﹚法测定体脂率;
3)采用体脂率间接测量法测量体脂率:利用皮褶厚度计测量受试对象的臂部、肩胛部、腹部、髂部、大腿部5个部位皮褶厚度。
5.根据权利要求4所述的肥胖症疾病大鼠动物模型的能量代谢与体脂率评价,其特征是,所述食物利用率计算公式为:食物利用率=体重增长量/(喂食量-撒食量)。
6.根据权利要求4所述的肥胖症疾病大鼠动物模型的能量代谢与体脂率评价,其特征是,所述和Lees指数计算公式为:Lees指数=体重(g)×103/体长(cm) 。
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