CN114592011B - 一种ptdss2条件性基因敲除小鼠模型的构建方法 - Google Patents
一种ptdss2条件性基因敲除小鼠模型的构建方法 Download PDFInfo
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Abstract
本发明涉及一种使用CRISPR/Cas9技术构建PTDSS2基因条件性敲除小鼠模型的方法，其包括：1、设计针对鼠源PTDSS2基因Exon2‑Exon3的sgRNA，2、利用体外转录技术获得上述sgRNA，3、将打靶载体、上述sgRNA、Cas9蛋白共注射或共电转至小鼠受精卵等步骤。与传统ES打靶小鼠模型相比，本发明首次构建的PTDSS2基因条件性敲除小鼠模型具有高效、快捷、简便、成本便宜等特点，且可以通过Cre来调控基因在特定的组织或特定时间进行表达。
Description
技术领域
本发明属于基因工程领域，具体涉及一种PTDSS2条件性基因敲除小鼠模型的构建方法及其应用。
背景技术
CRISPR/Cas9技术是通过设计特异性sgRNA实现特异性的DNA识别，并且在靶向位置完成切割，再通过细胞本身的DNA修复机制，完成断裂处的修复，从而实现目标基因的“编辑”。而随着CRISPR/Cas9技术的发展和研究的深入，其被广泛应用于构建转基因的模式动物（主要为基因编辑小鼠），从而为临床前提供动物层面治疗的基础研究。
人类疾病的发病机制研究和筛选有效的治疗药物均需要进行大量临床前试验。由于直接利用人细胞及组织进行临床前相关研究受到伦理方面的限制，动物模型就成为人类生物学研究的替代选择。小鼠具有体积小、易维持与操作、繁殖周期短、与人在基因组和生理学等特征方面相似，以及已经有相应成熟的基因修饰技术等优势，因此成为广泛应用的哺乳动物模式生物系统。但由于小鼠生理特征与人存在较多差异，利用动物模型得到的实验结果有时不能适用到人体上。然而利用基因修饰或细胞与组织移植的方法，将人类基因或细胞组织“放置”在小鼠模型上所制备的人源化小鼠模型（即带有人类功能性基因、人类细胞或组织的小鼠模型），极大程度上模拟人类基因或细胞组织的相关活动，大大提高了这类小鼠模型作为模拟某些人类疾病的有效性。
PTDSS2（Phosphatidylserine Synthase 2）是一种蛋白质编码基因，该基因编码的蛋白质能够催化磷脂酰乙醇胺转化为磷脂酰丝氨酸。磷脂酰丝氨酸是一种结构膜磷脂，在细胞信号传导、凝血和凋亡中起作用，该基因的一个重要旁系同源物是PTDSS1。目前关于PTDSS2机制研究比较少，CKO小鼠模型有利于进行相应分子以及功能等方面的研究。
PTDSS2 Cas9-CKO小鼠是利用基因修饰的方法，在小鼠的PTDSS2基因上的特定位点插入LoxP，将flox小鼠与表达Cre重组酶的小鼠交配，利用Cre-LoxP系统获得PTDSS2基因条件性敲除小鼠，同时通过Cre诱导可以实现在特定的组织或者特定时间对PTDSS2基因的敲除。
目前暂未检索到采用CRISPR/Cas9技术构建PTDSS2基因条件性敲除小鼠的相关报道。
发明内容
为解决上述技术问题，本发明包括以下几个方面：
本发明的第一方面提供一种PTDSS2基因条件性敲除小鼠模型构建方法，该方法包括如下步骤：
（1）设计针对鼠源PTDSS2基因Exon2-Exon3的sgRNA；
（2）利用体外转录技术获得上述sgRNA；
（3）将打靶载体、步骤（2）获得的sgRNA、Cas9蛋白共注射或共电转至小鼠受精卵细胞质或细胞核中，并将该受精卵移植至假孕小鼠，对假孕生仔鼠进行基因型鉴定，筛选成功插入正确人源片段的阳性F0小鼠；
（4）F0小鼠与背景鼠配繁获得F1小鼠，对F1代鼠尾进行基因鉴定，筛选出PTDSS2基因条件性敲除小鼠模型。
优选的，所述步骤（1）中两端sgRNA识别位点分别位于小鼠PTDSS2基因的第二个外显子之前和第三个外显子之后。
优选的，所述步骤（1）中sgRNA的基因序列为SEQ ID NO.1、SEQ ID NO.2、SEQ IDNO.3和SEQ ID NO.4中的一个或多个。
更优选的，所述步骤（1）中sgRNA的基因序列为SEQ ID NO.1和SEQ ID NO.2。
优选的，所述步骤（2）中以sgRNA-F、sgRNA-R为引物对，puc57-sgRNA质粒为模板进行PCR，PCR产物纯化制备sgRNA转录模板，并经过体外转录得到sgRNA。
优选的，所述步骤（2）中的引物对选自SEQ ID NO.5和SEQ ID NO.6，SEQ ID NO.7和SEQ ID NO.8，SEQ ID NO.9和SEQ ID NO.10，SEQ ID NO.11和SEQ ID NO.12所示序列对中一对或多对。
更优选的，所述步骤（2）中的引物对如SEQ ID NO.5和SEQ ID NO.6，以及SEQ IDNO.7和SEQ ID NO.8所示。
优选的，所述步骤（2）中的PCR反应体系如下：
优选的，所述步骤（2）中的PCR反应条件如下：
优选的，所述步骤（3）中提供受精卵的小鼠和假孕小鼠的品系为C57BL/6J。
优选的，所述步骤（3）中F0小鼠基因型鉴定使用的5’端鉴定引物如SEQ ID NO.13和SEQ ID NO.14所示，3’端鉴定引物如SEQ ID NO.15和SEQ ID NO.16所示。
优选的，所述步骤（3）中F0小鼠基因型鉴定使用的PCR反应条件如下：
优选的，所述步骤（3）中F0小鼠基因型鉴定使用的PCR反应体系如下：
本发明的第二方面提供模型构建方法得到的小鼠在研究PTDSS2基因相关功能和作用机制中的应用。
优选的，所述应用是非诊断和非治疗目的的。
本发明的第三方面提供上述构建方法得到的小鼠在筛选用于治疗与PTDSS2基因相关疾病的药物中的应用。
优选的，所述应用是非诊断和非治疗目的的。
本发明产生的有益效果：
与传统ES打靶小鼠模型相比，本发明的PTDSS2基因条件性敲除小鼠模型具有高效、快捷、简便、成本便宜等特点，且CKO模型可以通过Cre来调控基因在特定的组织或特定时间进行表达，可以满足小鼠模型在不同研究方向的需求，同时节省了时间及成本。
附图说明
图1为本发明F0代小鼠PTDSS2-CKO 5端、3端与野生型鉴定电泳图（DNA Marker条带：600bp\500bp\400bp\300bp\200bp\100bp）；
图2为本发明F1代小鼠PTDSS2-CKO 5端、3端与野生型鉴定电泳图（DNA Marker条带：600bp\500bp\400bp\300bp\200bp\100bp）；
图3为本发明小鼠PTDSS2-CKO 5端、3端与野生型鉴定电泳图中DNA Marker条带示意图。
具体实施方式
实施例1、PTDSS2基因条件性敲除小鼠模型的构建
本实施例使用CRISPR Cas9技术对小鼠PTDSS2基因进行基因修饰，基因稳定遗传的小鼠与表达Cre重组酶的小鼠交配后，flox小鼠被敲除，敲除该区域将导致蛋白质功能被破坏。如无特别说明，本实施例采用的小鼠均为C57BL/6J品系小鼠。
1、确定条件性敲除区域及序列
根据PTDSS2基因的结构，将PTDSS2-201转录本的Exon2-Exon3作为敲除区域，该区域包含185bp的编码序列。打靶载体的序列如下所示，其中具有下划线的大写字母对应Loxp位点。
Loxp1的插入位置如下所示：
cttgaagacctgacctgggaccggccctgtccctttcttggcaacgccttcatggttccctggttggttctggcagggttgccttcctacccactcagcgatttgtcacgtcctgcacgacttaagggatccagatctgggcccacaATAACTTCGTATAGCATACATTATACGAAGTTATatgttcttgcgcaaggtcagttggtgaggagcgtggtcatctccagggaagaacaggaactgggtattttccactcagagcaaggaaccagtttgtcctcatgcctgcccctgggcagtaactg
Loxp2的插入位置如下所示：
tgctttgtcacaataaaactacatgtgtacagggacaggtgctggctgacgggtaagggcttgtcctgcatttggaggaagagagctataagcccatatgatcgcattgtctgagtacgtgATAACTTCGTATAGCATACATTA TACGAAGTTATgggccaacagaattcgatatcctgcagggactaacagaagaacgcgttgtggtaaggcaacttcctctcaagagccacatctcaaggagcatgacagatgggaccacactggccaactggcagtaacttgcttctgcaacttggctccggg
2、确定用于模型制备的sgRNA序列
根据替换片段确定sgRNA大概区域，针对目的片段选取脱靶率最低几组序列作为待选sgRNA（见下表1）。设计并合成识别5’端靶位点和3’端靶位点，并构建sgRNA表达载体。两端sgRNA识别位点分别位于小鼠PTDSS2基因的第二个外显子之前和第三个外显子之后，各sgRNA在PTDSS2上的靶位点序列（SEQ ID NO.1至SEQ ID NO.4）如下：
表1 sgRNA序列信息
sgRNA转录制备方法：以PrimerStar或PrimerStar Max体系，sgRNA-F、sgRNA-R为引物，测序正确的puc57-sgRNA质粒（1：30稀释）为模板进行PCR（PCR反应体系和反应条件如表3和表4所示），PCR产物进行纯化，制备sgRNA转录模板。使用T7-ShortScript体外转录试剂盒（AM1354）进行sgRNA的转录。
sgRNA筛选：分别将4个sgRNA与Cas9蛋白进行孵育后，将混合液注射至0.5天的受精卵中，培养至囊胚阶段后，进行小鼠PTDSS2基因的KO阳性率的鉴定，以此筛选切割活性高的sgRNA。
sgRNA切割鉴定方法：对收集的囊胚进行巢式PCR扩增，PCR扩增引物序列见表2，将扩增条带进行二代测序，与wt条带比对，统计发生突变的概率，切割效率的鉴定结果见表5。
表2 sgRNA的PCR扩增引物序列
表3 PCR反应条件
表4 PCR反应体系
表5 sgRNA切割效率
3、打靶载体构建
注射用载体由通用生物（安徽）股份有限公司合成。
4、移植注射获得阳性鼠
将打靶载体、目的sgRNA（JS25721-Ptdss2-5S1和JS25721-Ptdss2-3S1）、Cas9蛋白注射到受精卵，移植至假孕小鼠。对假孕生仔鼠（1#-13#）进行基因型鉴定，鉴定引物信息见表6，PCR反应条件和反应体系如下表7和表8所示，筛选成功插入正确人源片段的阳性鼠F0小鼠，F0鉴定结果如图1所示，其中1#、4#、10#、13#均为阳性鼠F0，使用1#阳性鼠F0继续繁育。
表6 鉴定引物信息
表7 PCR反应条件
表8 PCR反应体系
F0小鼠（1#）与背景鼠配繁获得F1，对F1代鼠尾进行基因鉴定，F1代小鼠（23#-25#）的PCR实验结果如图2所示，其中23#为阳性鼠F1。阳性鼠F1大量扩繁后进行互配，获得纯合子。
以上所述的具体实施方式，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施方式而已，并不用于限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
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Claims (3)
1.一种PTDSS2基因条件性敲除小鼠模型构建方法，其特征在于，该方法包括如下步骤：
（1）设计针对鼠源PTDSS2基因Exon2-Exon3的sgRNA；
（2）利用体外转录技术获得上述sgRNA；
（3）将打靶载体、步骤（2）获得的sgRNA、Cas9蛋白共注射或共电转至小鼠受精卵细胞质或细胞核中，并将该受精卵移植至假孕小鼠，对假孕生仔鼠进行基因型鉴定，筛选成功插入正确人源片段的阳性F0小鼠；
（4）F0小鼠与背景鼠配繁获得F1小鼠，对F1代鼠尾进行基因鉴定，筛选出PTDSS2基因条件性敲除小鼠模型；
所述步骤（1）中两端sgRNA识别位点分别位于小鼠PTDSS2基因的第二个外显子之前和第三个外显子之后；
所述步骤（1）中sgRNA的基因序列为SEQ ID NO.1和SEQ ID NO.2。
2.根据权利要求1所述的小鼠模型构建方法，其特征在于，所述步骤（2）中以sgRNA-F、sgRNA-R为引物对，puc57-sgRNA质粒为模板进行PCR，PCR产物纯化制备sgRNA转录模板，并经过体外转录得到sgRNA；
所述步骤（2）中的引物对如SEQ ID NO.5和SEQ ID NO.6，以及SEQ ID NO.7和SEQ IDNO.8所示。
3.根据权利要求1所述的小鼠模型构建方法，其特征在于，所述步骤（3）中F0小鼠基因型鉴定使用的5’端鉴定引物如SEQ ID NO.13和SEQ ID NO.14所示，3’端鉴定引物如SEQ IDNO.15和SEQ ID NO.16所示。
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