CN111304252A - 基于pink1及park7的非治疗目的的将病毒注入动物特定脑区进行基因编辑的方法 - Google Patents
基于pink1及park7的非治疗目的的将病毒注入动物特定脑区进行基因编辑的方法 Download PDFInfo
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Abstract
本发明公开了一种基于PINK1及PARK7的非治疗目的的将病毒注入动物特定脑区进行基因编辑的方法，利用脑立体定位注射泵将病毒直接注射入非人灵长类动物的特定脑区域中；注射量为每1m3注射病毒1uL，注射病毒的滴度为10*1013vg/mL。该方法直接对非人灵长类动物的特定脑区进行基因编辑，可以短时间内获得具有疾病表型的转基因猴模型，解决现阶段迫切的帕金森氏综合征(PD)的研究需求。
Description
技术领域
本发明属于非人灵长类动物的转基因技术及建模领域，具体涉及一种基于 PINK1及PARK7基因(注：PARK7与DJ-1为同一基因)的非治疗目的的将病毒注入非人灵长类动物特定脑区进行基因编辑的方法，从而构建非人灵长类动物 PD疾病模型。
背景技术
帕金森病(Parkinson’s disease，PD)是典型的神经退行性疾病。该病病因及机制不明，主要在65岁以上的老年群体中发病，目前尚无治愈之法，在给患者造成巨大痛苦的同时，也给广大家庭与社会的带来沉重负担。由于在进化上高度相近，非人灵长目动物模型是目前最适合研究PD发病机制与研发有效治疗手段的模型之一。
虽然经典的MPTP猕猴模型已经广泛应用于PD研究的多方面，但是因为它是由化学药物诱导而成，不是一个病因型模型，因而在探讨PD的发病原因以及病变机制上基本无能为力。
发明内容
针对现有技术的不足，本发明提供了一种基于PINK1及PARK7基因的非治疗目的的将病毒注入非人灵长类动物特定脑区进行基因编辑的方法。
本发明所采用的技术方案：一种基于PINK1及PARK7基因的非治疗目的的将病毒注入非人灵长类动物特定脑区进行基因编辑的方法，用微量注射器将病毒直接注射入非人灵长类动物的特定脑区域中；注射量为每1m3注射病毒1uL，注射病毒的滴度为10*1013vg/mL。
进一步地，上述非人灵长类动物为猕猴或食蟹猴；所述非人灵长类动物的特定脑区域为单侧或双侧黑质脑区。所述可表达靶向猕猴PINK1和PARK7基因的sgRNA如序列表中所示。
进一步地，上述病毒以600～800nL/min的速度进行注射，注射后留针 10～15min。所述病毒为腺相关病毒(adeno-associated virus,AAV)。进一步地，上述微量注射器放置在立体定位仪的注射泵上，在MRI的定位指导下利用多点阵列注射法进行注射。
本发明具有以下显著特点：有效避免非人灵长类个体间的遗传差异和胚胎转基因技术嵌合体问题，以及传统胚胎转基因技术的死亡率高、成功率低、转入基因保留的稳定性差，成模时间长等不足。直接将病毒注入非人灵长类动物特定脑区进行基因编辑，可以短时间内获得转基因猴模型，解决现阶段迫切的帕金森氏综合症(PD)的研究需求。
附图说明
图1是本发明实施例所述基于PINK1及PARK7基因的非治疗目的的将病毒注入非人灵长类动物特定脑区的注射原理示意图。
图2是本发明实施例中PINK1及PARK7基因编辑方法的原理示意图。
具体实施方式
猕猴与人有良好的亲缘性、十分相似的脑结构、其基因与人类基因相似度高达93％(Huang,Zhang and Chen 1993)。另外，猕猴具有推理、分析、决策、记忆等高级脑功能，能完成除人外最复杂的行为学任务，相比其他动物也更容易进行复杂和精确的行为学检测。基于此，我们拟开发以AAV病毒为载体利用 CRISPR/CAS9技术突变猕猴PINK1及PARK7基因，以MRI成像为精确指导，特异地将该病毒注射到猕猴的单侧或双侧黑质内，建立病毒转基因PD猕猴模型。然后采用公认的猕猴PD行为量表(Kurlan scale)及阿扑吗啡诱导的旋转行为来评定模型动物的PD行为症状，并进行免疫组织化学病理学验证，希望获得高度类似人类PD患者的转基因PD猕猴模型，为探索该病的病因、病程、发病机制和开发新药供有效的猕猴模型。
因此，本发明的实施例提出一种直接将AAV注入非人灵长类动物脑区特定脑区进行基因编辑的建模方法，以下以猕猴为例进行说明。
如图1所示，本实施例采用直接向猕猴的特定脑区域注射腺相关病毒(AAV)，利用CRISPR/Cas9基因编辑技术敲除注射脑区猕猴的PINK1及PARK7基因，构建出能模拟PD核心症状的转基因猕猴。具体操作步骤：
(1)在病毒注射前，在猕猴生活的猴群中利用采用公认的猕猴PD行为量表(Kurlan scale)及阿扑吗啡诱导的旋转行为来评定动物的基础行为。
(2)利用MRI(核磁共振成像)深部脑结构精确定位技术，定位注射的特定脑区域，本实施例中为单侧或双侧黑质脑区。在MRI的定位指导下，利用多点阵列注射法将AAV病毒精确地注射入猕猴的特定脑区域中。
具体地，在本实施例所述转基因时的方法：首先麻醉猕猴，逐层打开头皮及头皮表组织，在定位处颅骨钻孔；然后采用微量注射器抽取病毒，放置在立体定位仪的注射泵上，以600～800nL/min的速度进行注射(优选800nL/min的注射速度)，注射后留针10～15min(分钟)，注射量为每1m3注射病毒1uL，注射病毒的滴度在10*1013vg/mL。所述转基因用的病毒载体为AAV(腺相关病毒)。
(3)注射病毒后，对猕猴重新进行行为学采集及分析，通过与之前采集的基础行为对比，同时与空载病毒注射对照组猕猴进行比较，观察猕猴在转基因前后出现了哪些行为学上的改变，是否有PD疾病的症状出现。在取得行为学数据之后，处死部分猕猴进行海马及周边脑区组织学及基因突变的检测，并利用组化检测多巴胺神经元及PD病理标志物。
另外，本发明的实施例中，所述非人灵长类动物也可以选择食蟹猴，所述方法为CRISPR/Cas 9技术敲除PINK1及PARK7基因，所述动物模型为人类PD的转基因非人灵长类模型。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和修饰，这些改进和修饰也应视为本发明的保护范围。
Claims (7)
1.一种基于PINK1及PARK7基因的非治疗目的的将病毒注入非人灵长类动物特定脑区进行基因编辑的方法，其特征在于，用微量注射器将AAV病毒直接注射入非人灵长类动物的特定脑区域中；注射量为每1m3注射病毒1uL，注射病毒的滴度为10*1013vg/mL。
2.根据权利要求1所述基于PINK1及PARK7基因的非治疗目的的将病毒注入非人灵长类动物特定脑区进行基因编辑的方法，其特征在于，所述非人灵长类动物的特定脑区域为单侧或双侧黑质脑及区。
3.根据权利要求2所述基于PINK1及PARK7基因的非治疗目的的将病毒注入非人灵长类动物特定脑区进行基因编辑的方法，其特征在于，所述非人灵长类动物为猕猴或食蟹猴。
4.根据权利要求2所述基于PINK1及PARK7基因的非治疗目的的将病毒注入非人灵长类动物特定脑区进行基因编辑的方法，其特征在于，所述可表达靶向猕猴PINK1及PARK7基因的sgRNA如序列表中所示。
5.根据权利要求1-3中任一权利要求所述基于PINK1及PARK7基因的非治疗目的的将病毒注入非人灵长类动物特定脑区进行基因编辑的方法，其特征在于，所述病毒以600～800nL/min的速度进行注射，注射后留针10～15min。
6.根据权利要求5所述基于PINK1及PARK7基因的非治疗目的的将病毒注入非人灵长类动物特定脑区进行基因编辑的方法，其特征在于，所述病毒为腺相关病毒(adeno-associated virus,AAV)。
7.根据权利要求1所述基于PINK1及PARK7基因的非治疗目的的将病毒注入非人灵长类动物特定脑区进行基因编辑的方法，其特征在于，所述微量注射器放置在立体定位仪的注射泵上，在MRI的定位指导下利用多点阵列注射法进行注射。
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