CN103642836A - 一种基于crispr基因敲除技术建立脆性x综合症灵长类动物模型的方法 - Google Patents
一种基于crispr基因敲除技术建立脆性x综合症灵长类动物模型的方法 Download PDFInfo
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Abstract
一种基于CRISPR基因敲除技术建立脆性X综合症灵长类动物模型的方法，包括以下步骤：(1)建立FMR1基因敲除食蟹猴模型；(2)食蟹猴动物模型的鉴定及相关功能分析；(3)食蟹猴动物模型的神经特征和学习记忆能力测试。本发明用CRISPR基因敲除技术建立脆性X综合症疾病灵长类动物模型，填补了灵长类动物模型的空白，可以有效地模拟人类疾病的病理过程，作为研究人类疾病的最佳模式生物，有效地预测新疫苗、新药和新诊断试剂等在临床应用中的效果，大大降低新药研发的风险。
Description
技术领域
本发明涉及生物医学领域，具体为一种基于CRISPR基因敲除技术建立脆性X综合症灵长类动物模型的方法。
背景技术
脆性X染色体综合症（Fragile X syndrome, FXS）是X染色体连锁的一种最常见遗传性弱智，该病影响着约1/3600的男性和1/4000-6000的女性。病因是FMR1基因发生CGG高拷贝动态突变，导致FMR1基因甲基化（DNA methylation）和基因沉默 (gene silencing)，智力迟钝蛋白（FMRP）不表达或表达量降低导致。
有关FXS发病机理的知识主要来自对FMR1基因敲除小鼠模型的研究。FXS小鼠模型表现出部分与人类患者相似的临床症状，如：颤抖、运动共济失调、注意力缺陷、智力障碍。在分子水平，由于FMRP表达量减少或缺失，FXS神经元突触传递的相关蛋白表达量上调；在细胞水平，神经元突触的可塑性和发育受到影响，突触数量减少，树突变长、变细，树突基部的突起减少；促代谢型谷氨酸受体（metabotropic glutamate receptor, mGluR）信号相关的长时程抑制（long-term depression, LTD）受到抑制。小鼠动物模型的建立使得对FXS的研究具有可操作性、重复性和可比性，有助于认识该疾病的发生机制。但是由于种系之间的差异，FXS小鼠动物模型的研究不能完全代替高等动物模型的研究，特别是在神经药理和毒性测试方面。因此建立灵长类FXS疾病模型，研究神经发育动态过程中出现的疾病表型变化，对阐明FXS发病机制非常必要。
人类疾病的动物模型是疾病机理研究和新药研发的重要基础。长期以来，生物医学基础研究主要依赖于啮齿类动物模型。但由于啮齿类动物与人类之间存在巨大的种属差异，使得基础研究成果不能有效地转化为临床应用。灵长类动物作为人类的近亲，其组织结构、免疫、生理、代谢和生殖等方面与人类高度相似，利用其创建的人类疾病模型，能比其他模式动物更好地复制人类传染性和非传染性疾病模型，并有效地模拟人类疾病的病理过程，可作为研究人类疾病的最佳模式生物。同时，灵长类动物研究成果能直接转化为临床应用，能更有效地预测新疫苗、新药和新诊断试剂等在临床应用中的效果，大大降低新药研发的风险。
基因定点修饰是研究基因功能的重要手段之一，也可被用于人类遗传性疾病的治疗，因此这类技术成为现代分子生物学的研究热点。Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas)9系统成功被改造为第三代人工核酸内切酶，与锌指核酸内切酶（zinc finger nucleases, ZFNs）和类转录激活因子效应物核酸酶（transcription activator-like effector nucleases, TALENs）一样可用于各种复杂基因组的编辑。目前该技术成功应用于人类细胞、斑马鱼和小鼠以及细菌的基因组精确修饰，修饰类型包括基因定点InDel突变、基因定点敲入、两位点同时突变和小片段的缺失。由于其突变效率高制作简单及成本低的特点，被认为是一种具有广阔应用前景的基因组定点改造分子工具。
在对FXS疾病的研究中，迄今国内外尚无灵长类动物模型的报道。
发明内容
本发明针对现有FXS疾病小鼠动物模型的不足，公开了一种用CRISPR基因敲除技术建立FXS灵长类动物模型的方法，以期阐明FXS的发病机制。
为实现上述目的，本发明提供如下技术方案：
一种基于CRISPR基因敲除技术建立脆性X综合症灵长类动物模型的方法，包括以下步骤：
(1) 建立FMR1基因敲除食蟹猴模型；
(2) 食蟹猴动物模型的鉴定及相关功能分析；
(3) 食蟹猴动物模型的神经特征和学习记忆能力测试。
所述建立FMR1基因敲除食蟹猴模型，具体包括以下步骤：
1）CRISPR靶向修饰基因载体的构建；
2）食蟹猴促排卵和体外受精；
3）食蟹猴受精卵的显微注射；
4）受精卵体外培养、植入受体及靶向基因修饰动物的培育。
所述食蟹猴动物模型的鉴定及相关功能分析，具体包括以下步骤：
1）对CRISPR靶向位点进行DNA测序鉴定食蟹猴动物模型的基因型；
2）利用实时定量PCR（real-time PCR）检测食蟹猴细胞中FMR1基因mRNA水平的降低；
3）通过免疫荧光染色检测食蟹猴细胞中FMRP蛋白的缺失；
4）观察食蟹猴心脏、肝、脾脏、肾脏、肌肉等重要器官或组织是否发生病变；
5）观察食蟹猴是否有FXS所特有的大睾丸症。
所述食蟹猴动物模型的神经特征和学习记忆能力测试，具体包括以下步骤：
1）利用电生理实验检测海马CA1的外势场（extracellular field potential）变化，从而验证由DHPG诱导的mGluR-LTD的变化；
2）研究食蟹猴的探索行为。
本发明与以往技术相比，具有以下优点：
本发明用CRISPR基因敲除技术建立脆性X综合症疾病灵长类动物模型，填补了灵长类动物模型的空白，可以有效地模拟人类疾病的病理过程，作为研究人类疾病的最佳模式生物，有效地预测新疫苗、新药和新诊断试剂等在临床应用中的效果，大大降低新药研发的风险。
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基因敲除技术建立脆性X综合症灵长类动物模型的方法，包括以下步骤：
(1) 建立FMR1基因敲除食蟹猴模型；
步骤如下：
1）CRISPR靶向修饰基因载体的构建；
2）食蟹猴促排卵和体外受精；
3）食蟹猴受精卵的显微注射；
4）受精卵体外培养、植入受体及靶向基因修饰动物的培育；
(2) 食蟹猴动物模型的鉴定及相关功能分析；
步骤如下：
1）对CRISPR靶向位点进行DNA测序鉴定食蟹猴动物模型的基因型；
2）利用实时定量PCR（real-time PCR）检测食蟹猴细胞中FMR1基因mRNA水平的降低；
3）通过免疫荧光染色检测食蟹猴细胞中FMRP蛋白的缺失；
4）观察食蟹猴心脏、肝、脾脏、肾脏、肌肉等重要器官或组织是否发生病变；
5）观察食蟹猴是否有FXS所特有的大睾丸症；
(3) 食蟹猴动物模型的神经特征和学习记忆能力测试。
步骤如下：
1）利用电生理实验检测海马CA1的外势场（extracellular field potential）变化，从而验证由DHPG诱导的mGluR-LTD的变化；
2）通过一系列行为学实验，研究食蟹猴的探索行为，如：焦虑状况，声光惊吓，条件性恐惧，疼痛的敏感性，以及惊厥易感性等。
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 (4)
1.一种基于CRISPR基因敲除技术建立脆性X综合症灵长类动物模型的方法，其特征是，包括以下步骤：
(1) 建立FMR1基因敲除食蟹猴模型；
(2) 食蟹猴动物模型的鉴定及相关功能分析；
(3) 食蟹猴动物模型的神经特征和学习记忆能力测试。
2.根据权利要求1所述的一种基于CRISPR基因敲除技术建立脆性X综合症灵长类动物模型的方法，其特征是，所述建立FMR1基因敲除食蟹猴模型，具体包括以下步骤：
1）CRISPR靶向修饰基因载体的构建；
2）食蟹猴促排卵和体外受精；
3）食蟹猴受精卵的显微注射；
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基因敲除技术建立脆性X综合症灵长类动物模型的方法，其特征是，所述食蟹猴动物模型的鉴定及相关功能分析，具体包括以下步骤：
1）对CRISPR靶向位点进行DNA测序鉴定食蟹猴动物模型的基因型；
2）利用实时定量PCR（real-time PCR）检测食蟹猴细胞中FMR1基因mRNA水平的降低；
3）通过免疫荧光染色检测食蟹猴细胞中FMRP蛋白的缺失；
4）观察食蟹猴心脏、肝、脾脏、肾脏、肌肉等重要器官或组织是否发生病变；
5）观察食蟹猴是否有FXS所特有的大睾丸症。
4.根据权利要求1所述的一种基于CRISPR基因敲除技术建立脆性X综合症灵长类动物模型的方法，其特征是，所述食蟹猴动物模型的神经特征和学习记忆能力测试，具体包括以下步骤：
1）利用电生理实验检测海马CA1的外势场（extracellular field potential）变化，从而验证由DHPG诱导的mGluR-LTD的变化；
2）研究食蟹猴的探索行为。
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