CN113234756A - 一种基于CRISPR/Cas9技术的LAMA3基因敲除动物模型的构建方法 - Google Patents
一种基于CRISPR/Cas9技术的LAMA3基因敲除动物模型的构建方法 Download PDFInfo
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Abstract
本发明属于生物工程领域，涉及一种基于CRISPR/Cas9技术的LAMA3基因敲除动物模型的构建方法，包括靶向小鼠LAMA3基因的sgRNA载体构建、sgRNA的转录和纯化、F0代小鼠获取及鉴定、F1代杂合小鼠获取及鉴定、纯合后代的获取等过程构建LAMA3基因敲除动物模型，所构建的动物模型，解决了传统基因敲除技术中基因脱靶率高、动物成活率低的问题，通过筛选更为高效的靶向小鼠LAMA3基因的sgRNA载体，并对进一步降低脱靶率，提高小鼠后代阳性率及模型构建的成功率，使得本发明方法构建的动物模型在癌症转移机理研究及药物开发领域具有重要应用价值。
Description
技术领域
本发明属于生物工程领域，具体涉及利用基因修饰技术制作基因敲出动物模型的领域，更具体涉及一种基于CRISPR/Cas9技术的LAMA3基因敲除动物模型的构建方法。
背景技术
CRISPR/Cas系统是一种来源于细菌获得性免疫的由RNA介导Cas蛋白对目的基因进行靶向修饰的技术。经过研究者改造过的TypeⅡCRISPR/Cas9系统自2013年成功敲除哺乳动物细胞后，现在已经被应用于多种模式生物的基因敲除。CRISPR/Cas9系统载体构建简单快速，易操作，省时省力周期短，且几乎对所有物种都适用。CRISPR/Cas9和TALEN(Transcription Activator-like Effector Nucleases)的作用都是实现染色体上特定位点的双链断裂，然后引发自主损伤修复，修复会引发插入或缺失，从而造成基因序列永久的缺失，即基因敲除。针对每个基因，CRISPER/Cas9只需要构建一个sgRNA(singleguideRNA)，而且效率都很高，序列选择限制较小，只需要基因组上出现GG就可以。与zinc-finger nuclease(ZFN)和TALEN相比，CRISPER/Cas9引起的脱靶效应较高，但是使用成对sgRNA/Cas9-D10A>截短的sgRNA或者FoKI-dCas9可以极大的降低脱靶效应。目前，CRISPER/Cas9主要应用于基因定点突变(插入或缺失)、基因定点敲除、两位点同时突变、小片段的缺失、编码基因和非编码基因(lncRNA、microRNA)的靶向基因敲除。
层黏连蛋白LAMA3(Laminin Subunit Alpha 3)是细胞外基质分子，是构成基底膜的主要成分，和Ⅳ型胶原、纤维连接蛋白等一起构成基底膜。发明人所在研究团队发现层黏连蛋白可调节细胞转移和机械信号传导，约束肿瘤细胞的侵袭和转移。层黏连蛋白是由α、β、γ亚基通过卷曲螺旋域组装而成的异三聚体分子，LAMA3基因编码层黏连蛋白α亚单位，其变异影响层黏连蛋白的表达，与人类肿瘤的发生发展密切相关。LAMA3基因异常表达与癌症的发生、发展密切相关，可作为癌症早期诊断或评估患者风险的指标，并可根据其异常表达程度来评估癌症的发展阶段，制定合理的诊疗计划。构建LAMA3基因敲除小鼠模型，对于研究LAMA3作为靶向基因用于癌症的靶向基因治疗等具有重要意义,但传统的基因敲除方法成功率极低，一直未得到应用。近年来，CRISPR/Cas9技术得到广泛应用，为LAMA3基因敲除模型鼠的构建及其在肿瘤研究中的应用提供了可能性。因此，基于该前期研究成果构建LAMA3基因敲除的动物模型，对于癌症的风险评估及抑制癌转移的研究及药物开发具有极为重要的意义。
发明内容
本发明的目的在于提供一种简单、高效且稳定好的基于CRISPR/Cas9技术的LAMA3基因敲除动物模型的构建方法。
基于上述目的，本发明采用如下技术方案：
第一方面，本发明提供一种基于CRISPR/Cas9技术的LAMA3基因敲除动物模型的构建方法，包括以下步骤：
S1：靶向小鼠LAMA3基因的sgRNA载体构建
以LAMA3内含子位置作为敲除区域，根据靶基因LAMA3设计一对相应的sgRNA序列，sgRNA序列如SEQ ID NO.1和SEQ ID NO.2所示；
S2：sgRNA的转录和纯化
将步骤S1中合成的一对sgRNA和cas9 mRNA进行体外转录并进行纯化；
S3：F0代小鼠获取及鉴定
将步骤S2中获得的mRNA与cas9质粒一并通过显微注射的方式注入目标受精卵中，培育获得F0代小鼠；
将获得的F0代小鼠进行剪尾、genetyping鉴定，挑选LAMA3基因敲出的阳性F0代小鼠；
S4：F1代杂合小鼠获取及鉴定
将S3中鉴定为阳性的F0代雄性小鼠与野生型雌性小鼠于性成熟后配繁，将出生的F1代小鼠进行剪尾、genetyping鉴定，挑选LAMA3基因敲出的阳性F1代杂合小鼠；
S5：纯合后代的获取
将S4中阳性F1代杂合雌性小鼠、阳性F1代杂合雄性小鼠杂交，获得纯合后代，构建得到LAMA3基因敲除动物模型。
进一步地，sgRNA载体以PUC57-sgRNA载体为出发载体，含有针对LAMA3基因的sgRNA。
第二方面，本发明提供了一种由上述方法构建得到的LAMA3基因敲除动物模型。
第三方面，本发明提供了一种利用CRISPR/Cas9技术进行LAMA3基因敲除的试剂盒，该试剂盒包括sgRNA载体和用于检测LAMA3基因的检测试剂；sgRNA载体以PUC57-sgRNA载体为出发载体，含有针对LAMA3基因的sgRNA。
进一步地，上述试剂盒还包括用于表达Cas9mRNA的Cas9表达质粒。
第四方面，本发明提供了上述LAMA3基因敲除动物模型在癌症风险评估和抑制癌转移的药物开发中的应用。
与现有技术相比，本发明的有益效果如下：
本发明基于CRISPR/Cas9技术构建LAMA3基因敲除的动物模型，解决了传统基因敲除技术中基因脱靶率高、动物成活率低的问题，本发明筛选更为高效的靶向小鼠LAMA3基因的sgRNA载体，并对进一步降低脱靶率，提高小鼠后代阳性率及模型构建的成功率，在癌症转移机理研究及药物开发领域具有重要应用价值。
具体实施方式
实施例1
一种基于CRISPR/Cas9技术的LAMA3基因敲除动物模型的构建方法，包括以下步骤：
S1：靶向小鼠LAMA3基因的sgRNA载体构建
以LAMA3内含子位置作为基因敲除区域，根据靶基因LAMA3设计一对相应的sgRNA序列；BsaⅠ酶切pUC57-sgRNA载体，37℃水浴1h后，1％的琼脂糖电泳，回收酶切产物；然后将sgRNA引物进行退火；最后，连接退火产物与回收的酶切产物；然后将sgRNA引物进行退火；最后，连接退火产物与回收的酶切产物，转化大肠杆菌，挑选单克隆进行PCR，PCR结果呈阳性送测序验证，得到正确的sgRNA载体，其中sgRNA序列如下表1中的SEQ ID NO.1和SEQ IDNO.2所示。
表1基于LAMA3内含子位置靶基因设计sgRNA的序列
| sgRNA名称 | SEQ ID NO | 序列(5’to3’) |
| LAMA3-sgRNA1 | SEQ ID NO.1 | TCAAGTATGCTCGGTGAGCTTGG |
| LAMA3-sgRNA3 | SEQ ID NO.2 | TGTTCTACAATTGACCATAGTGG |
S2：sgRNA的体外转录
将步骤S1中合成的一对sgRNA和cas9 mRNA，利用转录试剂盒进行体外转录；转录试剂盒为AM1354+AM1908，购自Ambion公司；含有cas9 mRNA的Cas9表达质粒为cas9 DA0A(plasmid#42335)，Addgene。
S3：F0代小鼠获取及鉴定
(1)准备单细胞受精卵
于性成熟小鼠腹腔注射马绒毛膜促性腺激素，注射量为5IU/只，46-48小时后注射人绒毛膜促性腺激素，注射完人绒毛膜促性腺激素后将2只雌鼠与单放雌鼠合笼。第四天上午检栓，见栓的记为0.5天；脱颈椎处死见栓0.5天的小鼠，剪出输卵管，用显微镊取出成团的卵子，透明质酸酶消化后，挑选形态饱满，胞质均匀的受精卵于M16中培养。
(2)显微注射受精卵
将挑选的受精卵转移入准备好的M2条带中，排成一列(每列30-50枚左右)，共注射180枚。将注射皿放在倒置显微镜的载物台上，使M2液滴长条的方向与操作者垂直，即位于y轴上。将注射管刺入胞浆内，注入步骤S2中获得的mRNA与cas9质粒即Cas9 sgRNA体系，见到胞质松散后迅速退针。注射结束后，将受精卵转移至含有M16培养液的培养皿中，放入37℃，5％二氧化碳培养箱恢复0.5-1.0小时。将受精卵移植到E0.5天假孕受体内。移植后大约19-21天出生F0代小鼠。
(3)F0代小鼠出生与鉴定
出生小鼠数量为7只，全部存活，将获得的F0代小鼠出生1周后进行剪尾、利用PCR进行genetyping鉴定，得到7只阳性F0代小鼠，毛色为黑色，性别为5雌2雄。
利用PCR进行genetyping鉴定的所用试剂及PCR程序如下表2、3和4所示。
表2 PCR反应体系
| 试剂 | 体积(微升) | 浓度 |
| 反应缓冲液(浓缩液，使用时作10倍稀释) | 2.5 | -- |
| 双蒸水 | 16.75 | -- |
| 上游引物 | 1 | 10微摩尔 |
| 下游引物 | 1 | 10微摩尔 |
| 镁离子溶液 | 2 | 25毫摩尔 |
| dNTPs | 0.5 | 10毫摩尔/样 |
| Taq DNA聚合酶 | 0.25 | 5酶活单位/微升 |
| 模板 | 1 | 100纳克/微升 |
表3 PCR进行genetyping鉴定过程中涉及到的引物
| 引物名称 | 引物序列 | 扩增片段长度 |
| LAMA3-seq-F | CAGTATTTTTGTGTTACCAACGGCA | 野生型：611bp |
| LAMA3-seq-R | ATACCCCAAACCCAGCCTTAGTG | 基因敲除：210bp |
表4 PCR进行genetyping鉴定的程序
S4：F1代杂合小鼠获取及鉴定
将S3中鉴定为阳性的F0代雄性小鼠与野生型雌性小鼠于8周龄性成熟后配繁，将出生的F1代小鼠于1周龄进行剪尾、genetyping鉴定，具体鉴定方法参照S3中的方法，挑选LAMA3基因敲出的阳性F1代杂合小鼠。
S5：纯合后代的获取
将S4中阳性F1代杂合雌性小鼠、阳性F1代杂合雄性小鼠杂交，获得纯合后代，构建得到LAMA3基因敲除动物模型。
由上述方法构建的LAMA3基因敲除动物模型列表如表5所示，可以看出，由本发明所述方法构建的LAMA3基因敲除动物模型，具有动物成活率高，在癌症风险评估、癌症转移机理研究及抑制癌转移药物开发具有重要应用价值。
表5小鼠模型构建过程中小鼠成活率及基因型
实施例2
本实施例提供了一种利用CRISPR/Cas9技术进行LAMA3基因敲除的试剂盒，该试剂盒包括sgRNA载体、表达Cas9mRNA的Cas9表达质粒和用于检测LAMA3基因的检测试剂；sgRNA载体以PUC57-sgRNA载体为出发载体，含有针对LAMA3基因的sgRNA。其中sgRNA的序列如SEQID NO.1和SEQ ID NO.2所示。
Claims (6)
1.一种基于CRISPR/Cas9技术的LAMA3基因敲除动物模型的构建方法，其特征在于，包括以下步骤：
S1：靶向小鼠LAMA3基因的sgRNA载体构建
以LAMA3内含子位置作为敲除区域，根据靶基因LAMA3设计一对相应的sgRNA序列，sgRNA序列如SEQ ID NO.1和SEQ ID NO.2所示；
S2：sgRNA的转录和纯化
将步骤S1中合成的一对sgRNA和cas9 mRNA进行体外转录并进行纯化；
S3：F0代小鼠获取及鉴定
将步骤S2中获得的mRNA与cas9质粒一并通过显微注射的方式注入目标受精卵中，培育获得F0代小鼠；
将获得的F0代小鼠进行剪尾、genetyping鉴定，挑选LAMA3基因敲出的阳性F0代小鼠；
S4：F1代杂合小鼠获取及鉴定
将S3中鉴定为阳性的F0代雄性小鼠与野生型雌性小鼠于性成熟后配繁，将出生的F1代小鼠进行剪尾、genetyping鉴定，挑选LAMA3基因敲出的阳性F1代杂合小鼠；
S5：纯合后代的获取
将S4中阳性F1代杂合雌性小鼠、阳性F1代杂合雄性小鼠杂交，获得纯合后代，构建得到LAMA3基因敲除动物模型。
2.根据权利要求1所述的构建方法，其特征在于，所述sgRNA载体以PUC57-sgRNA载体为出发载体，含有针对LAMA3基因的sgRNA。
3.一种由权利要求1或2所述方法构建得到的LAMA3基因敲除动物模型。
4.一种利用CRISPR/Cas9技术进行LAMA3基因敲除的试剂盒，其特征在于，所述试剂盒包括sgRNA载体和用于检测LAMA3基因的检测试剂；所述sgRNA载体以PUC57-sgRNA载体为出发载体，含有针对LAMA3基因的sgRNA。
5.根据权利要求3所述的试剂盒，其特征在于，所述试剂盒还包括用于表达Cas9mRNA的Cas9表达质粒。
6.权利要求3所述LAMA3基因敲除动物模型在癌症风险评估或抑制癌转移的药物开发中的应用。
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| CN114958907A (zh) * | 2021-12-31 | 2022-08-30 | 河南省人民医院 | 基于CRISPR/Cas9技术构建Bcl6基因超级增强子敲除动物模型的方法及应用 |
| CN115843747A (zh) * | 2022-12-02 | 2023-03-28 | 吉林大学 | 一种Lama3基因点突变的小鼠模型及其构建方法 |
| CN115896174A (zh) * | 2022-11-07 | 2023-04-04 | 浙江省中医院、浙江中医药大学附属第一医院（浙江省东方医院） | Gas5基因敲除小鼠构建肾纤维化的方法 |
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| CN114958907A (zh) * | 2021-12-31 | 2022-08-30 | 河南省人民医院 | 基于CRISPR/Cas9技术构建Bcl6基因超级增强子敲除动物模型的方法及应用 |
| CN115896174A (zh) * | 2022-11-07 | 2023-04-04 | 浙江省中医院、浙江中医药大学附属第一医院（浙江省东方医院） | Gas5基因敲除小鼠构建肾纤维化的方法 |
| CN115843747A (zh) * | 2022-12-02 | 2023-03-28 | 吉林大学 | 一种Lama3基因点突变的小鼠模型及其构建方法 |
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