CN107354173A - 基于crispr技术和水动力尾静脉注射建立肝脏特异性敲除小鼠模型的方法 - Google Patents

基于crispr技术和水动力尾静脉注射建立肝脏特异性敲除小鼠模型的方法 Download PDF

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CN107354173A
CN107354173A CN201611216919.XA CN201611216919A CN107354173A CN 107354173 A CN107354173 A CN 107354173A CN 201611216919 A CN201611216919 A CN 201611216919A CN 107354173 A CN107354173 A CN 107354173A
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tail vein
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mouse
crispr
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马月
余陈欢
应华忠
张欢欢
方杰
莫丽
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Zhejiang Academy of Medical Sciences
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Abstract

基于CRISPR技术和水动力尾静脉注射建立肝脏特异性敲除小鼠模型的方法，属于生物技术领域。该方法通过水动力尾静脉注射输送CRISPR系统在肝脏特异性表达建立肝脏特异性敲除小鼠模型。本发明不仅可以针对性使用CRISPR/Cas9系统进行肝脏特异性的模型研究，而且可以为肝脏重大疾病和新药研发提供高水平的动物模型。

Description

基于CRISPR技术和水动力尾静脉注射建立肝脏特异性敲除小 鼠模型的方法
技术领域
本发明属于生物技术领域，具体涉及基于CRISPR技术和水动力尾静脉注射建立肝脏特异性敲除小鼠模型的方法。
背景技术
CRISPR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(Terry Van Dyke , T.J,2002)。由于转基因动物传统制备的复杂性，近年来，具有序列特异性的核酸酶广泛应用于动物的基因组定点修饰，其中，ZFN 和TALEN 是两种最常见的基因编辑技术， ZFN 和TALEN 系统均基于蛋白质工程，构建基因(编码特异性结合靶基因的蛋白)较困难，而且由于切割时以二聚体的形式发挥作用，需要成对设计，技术难度较大，构建组装时间较长(Kim, H. and J.S. Kim, 2014)。而CRISPR/Cas系统，是细菌特有的一种获得性免疫系统，研究人员将其改造成为靶向基因组编辑的工具，由于操作简单、成功率高且效率高，成为了靶向基因组编辑工具中的佼佼者(Gasiunas, G., et al.2012.)。CRISPR/Cas 技术已经在基因功能研究、动物模型建立、基因治疗等领域得到广泛的推广和应用，国内外研究表明，应用CRISPR/Cas系统构建小鼠动物模型为全身性敲除。
水动力基因转移技术是一种简便、高效的体内基因转染方法，近年来已有成熟地发展。它是在高压下经小鼠尾静脉快速注射含目的基因重组质粒的生理盐水，从而在小鼠体内(主要在小鼠肝脏)实现目的基因的高效表达，常用于动物实验和实验动物的造模。
发明内容
针对现有技术存在的问题，本发明的目的在于设计提供一种基于CRISPR技术和水动力尾静脉注射建立肝脏特异性敲除小鼠模型的方法技术方案。
所述的基于CRISPR技术和水动力尾静脉注射建立肝脏特异性敲除小鼠模型的方法，其特征在于在AAV-GFP肝脏特异性表达小鼠模型中通过水动力尾静脉注射输送CRISPR系统px330-sgGFP建立肝脏特异性敲除小鼠模型。
所述的基于CRISPR技术和水动力尾静脉注射建立肝脏特异性敲除小鼠模型的方法，其特征在于所述的AAV-GFP肝脏特异性表达小鼠模型是AAV-GFP病毒液经尾静脉高压水动力注射的方法感染小鼠肝细胞而建立的小鼠模型。
所述的基于CRISPR技术和水动力尾静脉注射建立肝脏特异性敲除小鼠模型的方法，其特征在于所述的CRISPR系统px330-sgGFP由px330质粒的多克隆位点BasI插入sgGFP得到。
所述的一种肝脏特异性敲除小鼠模型，由任一上述记载的方法制备获得。
所述的一种肝脏特异性敲除小鼠模型，其特征在于所述的小鼠为雌性7-8 周龄FVB/NJ小鼠。
本发明与现有技术相比，具有以下优点：为了解决真核质粒转染的瞬时表达的问题，以及慢病毒转染体系病毒滴度低转染效率低下的问题，本发明特别利用 AAV-GFP病毒液通过小鼠尾静脉水动力注射建立肝脏特异性稳定表达GFP小鼠模型，达到稳定高效率的肝脏特异性表达目的基因。在此模型基础上，进一步使用CRISPR/Cas9系统进行GFP肝脏特异性敲除模型构建，建立小鼠肝脏特异性的快速基因敲除模型。CRISPR/Cas9基因编辑技术操作简单且效率高，但由于 CRISPR/Cas9系统敲除动物模型为全身性敲除，本发明利用高压尾静脉水动力技术输送CRISPR/Cas9系统特异性到动物肝脏表达，从而实现CRISPR/Cas9系统的靶向组织特异性敲除的功能。本发明不仅可以针对性使用CRISPR/Cas9系统进行肝脏特异性的模型研究，而且可以为肝脏重大疾病和新药研发提供高水平的动物模型。
附图说明
图1为pEGFP-N1(CMV-GFP)质粒结构示意图；
图2为AAV-GFP质粒结构示意图；
图3为AAV-GFP肝脏特异性表达成像检测；
图4为CRISPR/CAS9系统示意图；
图5为px330-sgGFP质粒结构示意图；
图6a、6b为注射AAV-GFP/px330-sgGFP肝脏特异性敲除表型观察，其中图6a.为注射3天(72h)后生理盐水组和AAV-GFP组肝脏特异性表达, 图6b.为注射AAV-GFP/px330-sgGFP肝脏特异性敲除。
图7 px330-sgPTEN-sgp53-Cas特异性敲除对肝组织病理的影响。
具体实施方式
以下通过实施例进一步说明本发明。
实施例1：真核表达载体pEGFP-N1的建立
材料及来源：质粒pEGFP-N1来源于CLONTECH(GenBank Accession #U55762 Catalog #6085-1)，由本室保存，pEGFP-N1(CMV-GFP)质粒结构如图1所示。质粒小提试剂盒购自Omega公司，质粒大提提取试剂盒购自Axygen 公司。
方法：活化质粒pEGFP-N1甘油菌，用质粒大提提取试剂盒获得大量真核表达EGFP质粒1mg。
实施例2：腺相关病毒表达载体AAV-GFP的建立
材料及来源：pAV-U6-GFP(AAV-GFP)质粒来自于维真生物，pAV-U6-GFP(AAV-GFP)质粒结构如图2所示。质粒小提试剂盒购自Omega公司，质粒大提提取试剂盒购自Axygen 公司。AAV-GFP病毒液购自于维真生物，滴度达到1×1013 V.G/ml。
构建方法：活化质粒pAV-U6-GFP菌液，用质粒大提提取试剂盒获得大量质粒1mg。使用pAV-U6-GFP(AAV-GFP)质粒以及辅助质粒Ad Helper Vector和AAV Rep/Cap Vector共转染293T细胞，纯化测定滴度为1×1013 V.G/ml。
实施例3：小鼠尾静脉水动力注射真核表达质粒pEGFP-N1和腺相关病毒AAV-GFP
材料及来源：动物SPF级FVB/NJ小鼠，7-8 周龄，雌性，体重18-22g，由浙江中医药大学动物中心提供，饲养于浙江省医学科学院动物中心二级动物房。饲养条件按照SPF 级动物标准执行。
试剂：经Axygen 公司质粒大量提取纯化试剂盒纯化的质粒pEGFP-N1 及病毒液AAV-GFP ；生理盐水(0.9％的NaCl溶液)。
仪器：1ml、2ml 及5ml 注射器。
方法：高压水动力尾静脉注射：雌性SPF 级FVB/NJ小鼠分成三组，每组6只，分别经高压水动力尾静脉注射生理盐水(control组)、50-80μg 质粒pEGFP-N1(真核质粒表达组)及1011IU/ml AAV-GFP病毒液(腺相关病毒表达组)。
具体操作如下：先将小鼠尾巴置于白炽灯泡下照射或热台上，使小鼠尾静脉扩张；5-8 秒内将2-2.5ml(相当于小鼠体重的8％-10％体积) 的质粒溶液匀速注射到小鼠体内，注射后室温条件下观察小鼠反应。
实施例4：小鼠肝脏特异性表达GFP模型成像检测
方法：各组小鼠注射后24h、72h、2周后，乙醚麻醉后进行活体成像和取肝脏成像观察GFP的表达情况。
结果显示：如图3所示，注射生理盐水的实验对照组(control组)肝脏不发出绿色荧光，无GFP蛋白表达；pEGFP-N1(真核质粒表达组)肝脏在72h发出绿色荧光， 2周后无GFP蛋白表达；AAV-GFP病毒液(腺相关病毒表达组)在72h、两周后均表达GFP蛋白，绿色荧光强于真核表达组且两周后GFP蛋白表达稳定。提示腺相关病毒AAV-GFP更优于真核质粒pEGFP-N1建立小鼠肝脏特异性表达模型。因此，小鼠肝脏特异性CRISPR敲除模型在腺相关病毒AAV-GFP感染模型的基础上进行。
实施例5：CRISPR系统px330-sgGFP的设计构建
材料及来源：CRISPR质粒选用 px330质粒体系(购自Addgene: Plasmid 42230)，由本室保存。质粒小提试剂盒购自Omega 公司，质粒大提提取试剂盒购自Axygen 公司。引物合成和测序于上海生物工程公司，内切酶购自NEB公司，连接酶购自Takara公司。
构建方法：px330质粒采用内切酶BbsI 插入目的基因片段序列，采用分子克隆技术构建px330-sgRNAs-Cas9质粒，px330-sgRNAs-Cas9的结构如图4所示。
设计合成sgGFP-F：CACCGGGGCGAGGAGCTGTTCACCG，sgGFP-R：CCCCGCTCCTCGACAAGTGGCCAAA，两条oligos进行退火形成DNA双链。退火形成的DNA与内切酶BbsI酶切纯化后的线性px330载体连接，转化，阳性克隆筛选和鉴定。测序鉴定正确的阳性克隆进行活化，大量抽提质粒准备尾静脉水动力注射。
实施例6：小鼠尾静脉水动力注射输送CRISPR系统
材料及来源：动物SPF级FVB/NJ小鼠，7-8 周龄，雌性，体重18-22g，由浙江中医药大学动物中心提供，饲养于浙江省医学科学院动物中心二级动物房。饲养条件按照SPF 级动物标准执行。
试剂：病毒液AAV-GFP及经Axygen 公司质粒大量提取纯化试剂盒纯化的质粒px330-sgGFP-Cas9；生理盐水(0.9％的NaCl 溶液)。
仪器：1ml、2ml 及5ml 注射器。
方法：高压水动力尾静脉注射：雌性SPF 级FVB/NJ小鼠分成三组，每组6 只，分别经高压水动力尾静脉注射生理盐水(Control组)、1011AAV-GFP病毒液 (GFP腺相关病毒表达组) 及50-80μg 质粒px330-sgGFP-Cas9 (如图5所示，sgGFP组)。
具体操作如下：先将小鼠尾巴置于白炽灯泡下照射或热台上，使小鼠尾静脉扩张；5-8 秒内将2-2.5ml(相当于小鼠体重的8％-10％体积) 的生理盐水或病毒溶液匀速注射到小鼠体内，注射后室温条件下观察小鼠反应；AAV-GFP病毒液注射72h后为第一次开始注射50-80μg px330-sgGFP-Cas9质粒溶液，第一次注射72h后，再次尾静脉水动力注射50-80μg px330-sgGFP-Cas9质粒溶液，第二次注射72h后，进行第三次尾静脉水动力注射50-80μgpx330-sgGFP-Cas9质粒溶液，一共注射等量px330-sgGFP-Cas9质粒溶液三次，第三次注射结束72h后，恰好AAV-GFP病毒液注射两周后，进行小鼠肝脏特异性敲除成像检测观察。
实施例8：小鼠肝脏特异性CRISPR敲除模型成像检测
方法：各组小鼠注射后3天、9天、12天、2周后，乙醚麻醉后进行活体成像和取肝脏成像观察GFP的表达情况；
结果显示：如图6所示，注射生理盐水的实验对照组(control组)肝脏不发出绿色荧光，无GFP蛋白表达； AAV-GFP病毒液(腺相关病毒表达组)在3天、9天、12天、两周后均表达GFP蛋白；质粒px330-sgGFP-Cas9 (sgGFP组)在9天、12天、两周后GFP蛋白的表达明显减弱，明显低于GFP特异性表达组。提示尾静脉水动力注射输送CRISPR系统(px330-sgGFP-Cas9)可以快速进行小鼠肝脏特异性敲除模型建立。
实施例9：CRISPR系统双敲PTEN+p53对小鼠肝功能的影响
方法：雌性SPF 级FVB/NJ小鼠分成三组，分别为正常对照组，阴性干扰组及基因敲除组。每组5只。除正常对照组外，于第1、4、7天，阴性干扰组小鼠尾静脉注射生理盐水，基因敲除组小鼠尾静脉注射50μg 质粒px330- sgPTEN-sgp53-Cas9。于末次注射后3天，各组随机取若干小鼠，均处死后取肝脏适量，4％中性福尔马林溶液固定，常规石蜡包埋切片，油红O染色，于显微镜下观察各组小鼠肝组织病理变化。结果显示：如图7所示，正常对照组小鼠及注射生理盐水组的小鼠肝细胞排列紧密，边缘清晰，胞质未见脂滴。AAV-GFP/px330-sgGFP肝脏特异性敲除组肝细胞脂肪变性，体积增大，含有大量的红染颗粒，细胞核被推向周边，脂质沉积明显增多，结构被破坏，肝细胞排列紊乱，提示尾静脉水动力注射输送CRISPR系统(px330- sgPTEN-sgp53-Cas9)可抑制肝细胞PTEN和p53基因表达，导致肝组织脂代谢紊乱。

Claims (5)

1.基于CRISPR技术和水动力尾静脉注射建立肝脏特异性敲除小鼠模型的方法，其特征在于通过水动力尾静脉注射输送CRISPR系统在肝脏特异性表达建立肝脏特异性敲除小鼠模型。
2.如权利要求1所述的基于CRISPR技术和水动力尾静脉注射建立肝脏特异性敲除小鼠模型的方法，其特征在于所述的AAV-GFP肝脏特异性表达小鼠模型是AAV-GFP病毒液经尾静脉高压水动力注射的方法感染小鼠肝细胞而建立的小鼠模型。
3.如权利要求1所述的基于CRISPR技术和水动力尾静脉注射建立肝脏特异性敲除小鼠模型的方法，其特征在于所述的CRISPR系统px330-sgGFP由px330质粒的多克隆位点BasI插入sgGFP得到。
4.一种肝脏特异性敲除小鼠模型，由权利要求1-3 任一权利要求所述方法制备获得。
5.如权利要求4所述的一种肝脏特异性敲除小鼠模型，其特征在于所述的小鼠为雌性7-8 周龄小鼠。
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