CN116497060A - 一种构建新型肌萎缩侧索硬化症模型的方法及模型 - Google Patents
一种构建新型肌萎缩侧索硬化症模型的方法及模型 Download PDFInfo
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Abstract
本发明提供一种构建新型肌萎缩侧索硬化症模型的方法及模型，利用SpRY‑CBE单碱基基因编辑技术进行ARPP21基因p.P521L位点突变构建一对寡聚核苷酸链。本发明提供的一种构建新型肌萎缩侧索硬化症模型的方法及模型通过对ARPP21基因P.P521L位点进行突变打靶，胚胎注射后进行胚胎移植，获得兔先天性高胰岛素血症模型，利用SpRY‑CBE基因编辑技术优势是没有识别PAM区域的序列限制，可以完成指定位点的突变顺利进行；克服了传统的CRISPR/Cas9基因编辑技术需要识别PAM区域为NGG碱基序列，大部分突变位点无法进行编辑；本发明所利用的SpRY‑CBE碱基编辑技术首次在家兔上进行应用。该兔疾病模型不但能够有助于基础医学研究及机制探究，同时也是SpRY‑CBE碱基编辑系统首次在家兔动物中进行应用。
Description
技术领域
本发明涉及生物技术领域，特别的为一种构建新型肌萎缩侧索硬化症模型的方法及模型。
背景技术
肌萎缩侧索硬化症(ALS)简称渐冻症，是一种渐进性、神经退行性疾病，著名物理学家霍金就患此病。该病影响大脑和脊髓中的运动神经元，造成运动神经元死亡，令大脑无法控制肌肉运动，肌肉也会因缺乏运动而萎缩。尚无有效的治疗方案。
ARPP21基因编码cAMP调节的磷酸化蛋白。编码的蛋白质在尾状核和小脑皮层中富集。而小鼠中的类似蛋白质可能参与调节基底神经节中多巴胺的作用。交替剪接导致多个转录本变异。2020年研究表明ARPP21基因P.P521L位点突变可导致肌萎缩侧索硬化症。然而前期单碱基基因编辑技术有PAM限制及设计要求，而这个突变位点序列限制而无法完成，因此目前针对该突变位点还没有合适的动物模型。
发明内容
本发明提供的发明目的在于提供一种构建新型肌萎缩侧索硬化症模型的方法及模型以解决上述问题。
为实现以上目的，本发明通过以下技术方案予以实现：一种构建新型肌萎缩侧索硬化症模型的方法及模型，
1)sgRNA表达载体的构建：
针对基因P.P521L位点设计1个sgRNA序列作用靶点，合成两条寡聚核苷酸链制备sgRNA，sgRNA的寡聚核苷酸链选取原则：选取突变碱基位置在5或6位的一条寡聚核苷酸链，其寡聚核苷酸为：
sgRNA-1-F：TAGGATATCCGGCGGTCTCTTTC；
sgRNA-1-R：AAACGAAAGAGACCGCCGGATAT；
两条合成的寡聚核苷酸链经退火形成双链，退火条件是：95℃5min后自然降至室温，分BbsⅠ限制性核酸内切酶对PUC57载体线性化，随后将酶切产物进行回收，并将退火的sgRNA连接到PUC57载体上，进而完成PUC57-sgRNA载体的构建，如序列表SEQNO.3所示。
酶切37℃3h，电泳跑胶后，使用普通DNA琼脂糖胶回收试剂盒进行回收；
2)SpRY-CBE mRNA的合成：SpRY-CBE表达质粒经酶切线性化，经酚氯仿抽提纯化后，溶于无核酸酶的水中；
酶切37℃3h，电泳跑胶后，使用普通DNA琼脂糖胶回收试剂盒进行回收；
3)受精卵的获取和显微注射：注射卵泡刺激素，之后注射人绒毛膜促性腺激素，获取受精卵，通过显微注射仪器将预混好SpRY-CBE mRNA/sgRNA混合物注射到细胞质中，其中SpRY-CBE mRNA浓度为150ng/μl，sgRNA浓度为30ng/μl；
4)受精卵的培养和发育：将显微注射的受精卵转移到培养液中，置于37℃恒温培养箱中培养，发育到桑椹胚时期时，用吸卵针将单个胚胎转移到离心管中。
本发明还提供了胚胎ARPP21基因敲除情况鉴定方法：
1、胚胎裂解：胚胎裂解试剂为NP40，裂解条件为：56℃，1h；95℃，10min；
2、DNA测序鉴定胚胎基因型突变情况：提取DNA，进行PCR，电泳鉴定，并进行DNA测序，得到基因型鉴定结果；
a、设计PCR引物如下：
上游引物：CCACCACCAACATTGAACATAC；
下游引物：GGCAGCTAACAGGAAGGATAC；
b、PCR反应体系如下：
c、PCR反应条件：
95℃预变性7min；94℃变性30s，58℃退火30s，72℃延伸40s；30个循环；72℃延伸5min；
3、PCR产物进行测序，测序结果在ARPP21基因引物设计的打靶位点出现完全突变或不完全突变的情况，选择位点完全突变或者不完全突变的样品则为基因突变。
本发明提供了一种构建新型肌萎缩侧索硬化症模型的方法及模型。具备以下有益效果：
本发明提供的一种构建新型肌萎缩侧索硬化症模型的方法及模型通过对ARPP21基因P.P521L位点进行突变打靶，胚胎注射后进行胚胎移植，获得兔先天性高胰岛素血症模型，利用SpRY-CBE基因编辑技术优势是没有识别PAM区域的序列限制，可以完成指定位点的突变顺利进行；克服了传统的CRISPR/Cas9基因编辑技术需要识别PAM区域为NGG碱基序列，大部分突变位点无法进行编辑；本发明所利用的SpRY-CBE碱基编辑技术首次在家兔上进行应用。该兔疾病模型不但能够有助于基础医学研究及机制探究，同时也是SpRY-CBE碱基编辑系统首次在家兔动物中进行应用。
附图说明
图1是本发明ARPP21 sgRNA的设计示意图；
图2是本发明PCR产物鉴定胚胎ARPP21基因突变情况的sanger测序图；
图3是显微注射后得到的F0代新生兔ALS模型图；
图4是F0代新生兔ALS模型基因型鉴定图；
图5是F0代新生兔ALS模型体重水平检测图；
图6是F0代新生兔ALS模型运动电位水平检测图；
图7是F0代新生兔ALS模型肌肉病理观察图；
图8是本发明基因序列表。
具体实施方式
下面，结合附图以及具体实施方式，对本发明做出进一步的描述：
一种构建新型肌萎缩侧索硬化症模型的方法及模型，利用SpRY-CBE碱基编辑器成功构建ARPP21基因位点p.P521L突变先天性高胰岛素性低血糖血症兔模型：
1、SpRY-CBE系统sgRNA设计和表达载体的构建：
针对在ARPP21基因p.P521L位点设计一对sgRNA序列作用靶点，合成两条寡聚核苷酸链用于制备sgRNA；该sgRNA的寡聚核苷酸链选取原则：选取突变碱基位置在5或6位的一条寡聚核苷酸链；合成的寡聚核苷酸经退火(95℃5min后自然降至室温)，连入经BbsⅠ酶切经回收PUC57-sgRNA表达载体，完成sgRNA载体构建，通过测序验证片段连接正确，进行克隆，扩大培养后提取质粒用于准备体外转录模板；
酶切37℃3h，电泳跑胶后，使用普通DNA琼脂糖胶回收试剂盒(购于天根公司，北京，中国)进行回收；
SpRY-CBE表达质粒(Addgene，实验室购买)，经酶切线性化，经酚氯仿抽提纯化后，溶于无核酸酶的水中作为模板，用于体外转录；SpRY-CBE mRNA的合成由试剂盒RNeasyMini Kit(Qiagen,No.74104)在体外作用T7RNA聚合酶来完成，sgRNA的体外合成由试剂盒MiRNeasy Mini Kit(Qiasgen,No.217004)在体外利用T7RNA聚合酶完成；
酶切37℃3h，电泳跑胶后，使用普通DNA琼脂糖胶回收试剂盒(购于天根公司，北京，中国)进行回收。
2、受精卵的获取和显微注射。
注射卵泡刺激素(FSH)，之后注射人绒毛膜促性腺激素(HCG)(购于宁波第二激素厂)，获取受精卵，通过显微注射仪器将预混好SpRY-CBE mRNA/sgRNA混合物注射到细胞质中(SpRY-CBE mRNA终浓度为150ng/ul，sgRNA终浓度为30ng/ul)。
3、受精卵的体外培养和发育。
将显微注射的受精卵转移到培养液中，置于37℃恒温培养箱中培养，发育到桑椹胚时期时，用吸卵针将单个胚胎转移到离心管中，用于后面实验。
胚胎ARPP21基因突变情况鉴定：
1)胚胎裂解
胚胎裂解试剂为NP40，裂解条件为：56℃1h；95℃10min；
2)DNA测序鉴定胚胎基因型突变情况
提取DNA，提取方法按照组织基因组提取试剂盒说明书进行操作(购于天根公司，北京，中国)，进行PCR，电泳鉴定，并进行DNA测序，得到基因型鉴定结果；
(1)胚胎裂解：胚胎裂解试剂为NP40，裂解条件为：56℃，1h；95℃，10min；
(2)DNA测序鉴定胚胎基因型突变情况：提取DNA，进行PCR，电泳鉴定，并进行DNA测序，得到基因型鉴定结果；
a、设计PCR引物如下：
上游引物：CCACCACCAACATTGAACATAC；
下游引物：GGCAGCTAACAGGAAGGATAC；
b、PCR反应体系如下：
c、PCR反应条件：
95℃预变性7min；94℃变性30s，58℃退火30s，72℃延伸40s；30个循环；72℃延伸5min；
(3)PCR产物进行测序，测序结果在ARPP21基因引物设计的打靶位点出现完全突变或者不完全突变的情况，选择位点完全突变或者不完全突变的样品则为基因突变。
实验例1
ARPP21基因ALS兔模型表型鉴定和基因型分析：
1)DNA测序鉴定ARPP21基因突变ALS病兔模型的基因型
提取出生兔模型组织DNA，提取方法按照组织基因组提取试剂盒说明书进行操作(天根，北京，中国)，进行PCR，核酸电泳鉴定，并进行DNA测序，得到基因型鉴定结果；
a、设计PCR引物如下：
上游引物：CCACCACCAACATTGAACATAC；
下游引物：GGCAGCTAACAGGAAGGATAC；
b、PCR反应体系如下：
c、PCR反应条件：
95℃预变性5min；95℃变性30s，58℃退火30s，72℃延伸30s；35个循环；72℃延伸5min；
2)兔模型体重检测
分别于出生后每两周测定正常兔与突变兔的体重水平，如图5所示，出生16周后ARPP21基因突变兔模型体重出现明显降低；
3)兔生理学分析
在第24周，我们利用肌电仪对ARPP21基因突变ALS病兔模型神经元电传导及表面肌电信号进行检测，如图6所示，ALS疾病模型兔左右两侧腓肠肌静息电位不稳定，存在正锐波和纤颤；符合人类肌萎缩侧索硬化症肌电图的临床症状。
图8中，SEQNO.1为兔ARPP21基因CDS区序列，SEQNO.2为兔ARPP21基因突变后序列，SEQNO.3为PUC57-sgRNA1序列，斜体大写下划线标注为sgRNA一条寡聚核苷酸链。
4)兔病理学分析
突变兔在生长过程中，出现死亡的个体，解剖观察肌肉病变情况，固定组织，做组织病理切片；如图7所示，可见ARPP21(p.P521L)兔存在肌束萎缩。
结论：本发明成功构建ARPP21基因P.P521L位点突变兔模型，其具有典型的肌萎缩侧索硬化症症状，与人类临床病例结果相一致，本发明构建的模型准确可靠。
最后应说明的是：以上仅为本发明的优选实施例而已，并不用于限定本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (2)
1.一种构建新型肌萎缩侧索硬化症模型的模型，其特征在于，利用SpRY-CBE单碱基基因编辑技术进行ARPP21基因p.P521L位点突变构建一对寡聚核苷酸链，其方法如下：针对ARPP21基因p.P521L位点突变设计1个sgRNA序列作用靶点，合成一对寡聚核苷酸链制备sgRNA，其寡聚核苷酸为：
sgRNA-1-F：TAGGATATCCGGCGGTCTCTTTC；
sgRNA-1-R：AAACGAAAGAGACCGCCGGATAT。
2.根据权利要求1所述的一种构建新型肌萎缩侧索硬化症模型的方法及模型，其特征在于：包括以下步骤：
1)sgRNA表达载体的构建：
针对ARPP21基因p.P521L位点设计1个sgRNA序列作用靶点，合成一对寡聚核苷酸链，两条合成的寡聚核苷酸链经退火形成双链，分BbsⅠ限制性核酸内切酶对PUC57载体线性化，随后将酶切产物进行回收，并将退火的sgRNA连接到PUC57载体上，进而完成PUC57-sgRNA载体的构建，酶切37℃3h，电泳跑胶后，使用普通DNA琼脂糖胶回收试剂盒进行回收；
2)SpRY-CBEmRNA的合成：
SpRY-CBE表达质粒经酶切线性化，经酚氯仿抽提纯化后，溶于无核酸酶的水中；酶切37℃3h，电泳跑胶后，使用普通DNA琼脂糖胶回收试剂盒进行回收；
3)受精卵的获取和显微注射：
注射卵泡刺激素，之后注射人绒毛膜促性腺激素，获取受精卵，通过显微注射仪器将预混好SpRY-CBEmRNA/sgRNA混合物注射到细胞质中；
4)受精卵的培养和发育：
将显微注射的受精卵转移到培养液中，置于37℃恒温培养箱中培养，发育到桑椹胚时期时，用吸卵针将单个胚胎转移到离心管中。
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