CN114606270B - 一种Mia3条件性基因敲除小鼠模型的构建方法 - Google Patents
一种Mia3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Mia3基因条件性敲除小鼠模型的方法，其包括：1、设计针对鼠源Mia3基因Exon2‑Exon7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Mia3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Mia3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。
Mia3编码黑色素瘤抑制蛋白3，是一种进化保守的内质网常驻跨膜蛋白。有研究表明，Mia3敲除显示软骨发育不良，导致胎儿矮化、外周水肿和围产期致死。缺乏Mia3的小鼠对于从软骨细胞，成纤维细胞，内皮细胞和壁画细胞中分泌许多胶原蛋白（包括胶原蛋白I，II，III，IV，VII和IX）是有缺陷的。这些细胞类型的胶原沉积是异常的，并且细胞外基质组成受到损害。这些变化与胶原蛋白的细胞内积累和强烈的未折叠蛋白质反应的诱导有关，主要是在发育中的骨骼内。软骨细胞成熟和骨矿化在Mia3-null胚胎中受到严重损害，导致侏儒症和新生儿致死。因此，Mia3在蛋白质分泌中的作用比以前意识到的要广泛得多，实际上它可能是高等生物体中所有胶原蛋白分子有效分泌所必需的。因此开发一种Mia3 Cas9-CKO小鼠模型能够用于评价与Mia3基因缺失相关的各类研究。
条件性基因敲除（CKO）是通过把两个LoxP位点插入到目的基因的一个或几个重要外显子的两端以制备出有两个floxed小鼠。该floxed小鼠在与表达Cre重组酶小鼠杂交之前，该基因表达正常；当floxed小鼠与组织特异性表达Cre酶的小鼠进行杂交后，可实现在特定的组织或细胞中敲除该基因，而在其它组织或细胞中该基因表达正常。
Mia3 Cas9-CKO小鼠是利用基因修饰的方法，在小鼠的Mia3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Mia3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Mia3基因的敲除。
目前暂未检索到采用CRISPR/Cas9技术构建Mia3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Mia3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Mia3基因Exon2-Exon7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Mia3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Mia3基因的第二个外显子之前和第七个外显子之后。
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Mia3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Mia3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。
优选的，所述疾病为心血管疾病、癌症中的一种或多种。
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Mia3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Mia3-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Mia3-CKO 5端、3端与野生型鉴定电泳图（N为空白对照，B6为阴性对照基因组DNA，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、Mia3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Mia3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Mia3基因的结构，将Mia3-202转录本序列的Exon2至Exon7部分作为敲除区域，该区域包含3557bp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。Loxp 1插入第二外显子（Exon 2）的上游，Loxp 2插入第七外显子（Exon 7）的下游。
其中，Loxp1的插入位置如下所示：
AGCCACAAATGAAACTGGCAGTGTTTACTGCCGCAAAGAAACAGTTGCAGCCACTTACAGGCAGTGCCTGTTAGTGGTTAGCTCAGGTTTAATTGACCCCATTTGTCACGTCCTGCACGACTTAAGGGATCCAGATCTGGGCCCACAATAACTTCGTATAGCATACATTATACGAAGTTATATGTTCTTGCGCAAGGTCAGTTGGTTGGGGATTCCTGGGCGATCTTCCTGGCTCCCTGTTATCCCCACAGTTAGCTATGTTTAAACCAGTCAGCACCTGCACCTGCATTTACACATCCACAAAC。
Loxp2的插入位置如下所示：
TACAGATTATATGGTGTCCAAAGACACATTATTACATTTTCATTCTGTTTTTTCTGAATACAATTTGTGTTTCAGATATACACACTCACCTCTGGAACCTATCGCATTGTCTGAGTACGTGATAACTTCGTATAGCATACATTA TACGAAGTTATGGGCCAACAGAATTCGATATCCTGCAGGGACTAACAGAAGAACGCGTTGTGTGGTCTTTTCCCTAAGATACCTTCCTTGGTTTTGCTTCTGCCAACTTACCTATTCTTGTTAATTTGGGTGAAATGGGACAAGGCTATGGCATTAGAGAAA。
2、确定用于模型制备的sgRNA序列
根据据敲除区域确定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Mia3基因的第二个外显子之前和第七个外显子之后，各sgRNA在Mia3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（表2），sgRNA-F、sgRNA-R为引物，测序正确的puc57-sgRNA质粒为模板进行PCR（反应条件见表3），PCR产物进行纯化，制备sgRNA转录制备模板。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Mia3基因的KO阳性率的鉴定，以此筛选切割活性高的sgRNA。
表2 sgRNA的PCR扩增体系
表3 sgRNA的PCR反应条件
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4，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4 sgRNA的PCR扩增引物序列
表5 sgRNA切割效率
3、打靶载体构建
Loxp1、Loxp2打靶载体的序列由通用生物（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（wJS05987-Mia3-5S1和wJS05987-Mia3-3S1）、Cas9蛋白注射到受精卵，移植至假孕小鼠。对假孕鼠生的后代（JS05987 5#-8#）进行基因型鉴定，鉴定引物信息见表6，PCR鉴定反应体系和反应条件见表7和表8，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。从对照Marker条带（图3）大小对照来看，5端408bp为正确插入，3端434bp为正确插入，两端同时插入为阳性F0，图1中6#为阳性F0，将6#小鼠继续配繁。
表6 鉴定引物信息
表7 PCR鉴定反应体系
表8 PCR鉴定反应条件
阳性F0小鼠6#与背景鼠配繁获得F1，对F1代鼠尾进行基因鉴定，F1代小鼠（24#、26#、27#、28#、30#）的PCR实验结果如图2所示，从对照Marker条带可以看出，5端出现408bp条带为正确插入，303bp为WT，3端434bp为正确插入，328bp为WT，因此F1代小鼠24#、26#、27#、28#、30#皆为阳性小鼠，获得的阳性F1代小鼠即为本发明构建的Mia3基因条件性敲除小鼠模型。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。
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Claims (3)
1.一种Mia3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Mia3基因Exon2-Exon7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Mia3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Mia3基因的第二个外显子之前和第七个外显子之后；
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SEQID NO.7和SEQ ID NO.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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