CN116941576A - 一种Foxg1 p.Tyr392X点突变小鼠模型的构建方法及其应用 - Google Patents
一种Foxg1 p.Tyr392X点突变小鼠模型的构建方法及其应用 Download PDFInfo
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Abstract
本发明提供了一种Foxg1p.Tyr392X点突变小鼠模型的构建方法及其应用，属于动物模型领域。该方法包括以下步骤：构建打靶载体；对sgRNA序列进行设计并制备成显微注射RNA；将Cas9、显微注射RNA、打靶载体注射到小鼠的受精卵，经发育形成F0代小鼠；将F0代小鼠与野生型小鼠交配获得F1代小鼠并对其进行基因型鉴定及Southernblot鉴定等，确认Foxg1基因点突变小鼠模型构建成功。本发明是基于临床患者突变位置和类型所构建的Foxg1点突变小鼠模型，能够比较贴切地重现真实疾病的发生和发展过程，对于个性化探讨FOXG1综合征的致病机理、治疗方法、药物筛选等方面具有重要意义。
Description
技术领域
本发明涉及一种Foxg1 p.Tyr392X点突变小鼠模型的构建方法及其应用，属于动物模型领域。
背景技术
叉头框转录因子FOXG1杂合突变导致FOXG1综合征，患者表现为发育迟缓、认知缺陷、社交障碍、运动障碍等自闭症核心症状。此类神经发育疾病严重影响患者身心健康，给患者家庭和社会带来了沉重的精神和经济负担。
人FOXG1基因位于14号染色体q12上，目前已鉴定出多种FOXG1突变，已有的数据表明FOXG1突变位点不同，患者临床症状也各不相同。具体而言，人FOXG1蛋白全长489个氨基酸，包含N端叉头结构域(FBD，181-275位氨基酸)，Groucho结合结构域(GBD，307-317位氨基酸)，JARID1B结合结构域(JBD，383-406位氨基酸)和C端结构域。其中JBD结构域可招募其他转录抑制因子(如JARID1B，一种去甲基化酶)与FOXG1共同调控下游靶基因。临床上，JBD到C端结构域的突变主要为无义突变(其比例约为3/4)。
FOXG1 p.Tyr400X无义突变的患者，表现出典型的自闭症样行为，具有社交障碍、语言障碍、运动障碍、眼神接触障碍和手部重复刻板行为等。由于物种差异，在小鼠中该突变位置对应的是Foxg1 p.Tyr392X。以往针对该位点突变的相关研究报道甚少，目前仅有一例并且是在细胞系中进行的实验。但从未有研究报道过Foxg1 p.Tyr392X在生物体内的功能，并且目前基因过表达和敲除小鼠均无法贴切地模拟临床患者的症状，无法体现出FOXG1蛋白在第392位酪氨酸处发生无义突变所导致的生物学功能。因此，Foxg1 p.Tyr392X无义突变小鼠模型的构建十分有必要。
发明内容
针对现有技术的不足，为了更加真实贴切的模拟FOXG1综合征患者的表型，本发明的目的在于提供一种Foxg1 p.Tyr392X点突变小鼠模型的构建方法及其应用。具体地，本发明的目的可以通过以下技术方案实现：
根据小鼠Foxg1未突变前基因序列，设计sgRNA序列；
按照所述设计的sgRNA序列合成oligos，所述oligos通过Gibson Assembly方法连入包含Cas9的pCS-4G载体；
设计引物构建打靶载体；
将Cas9、sgRNA和打靶载体显微注射到小鼠受精卵中，得到F0代小鼠，设计引物对F0代小鼠进行测序；
所述F0代小鼠与野生型小鼠交配，得到F1代小鼠，设计引物对F1代小鼠进行测序。
进一步，所述小鼠Foxg1未突变前基因序列包括SEQ ID NO.25所示序列。
进一步，所述sgRNA包括5’靶位点的SEQ ID NO.1～8所示序列和3’靶位点的SEQID NO.9～16所示序列。
优选地，所述5’靶位点的sgRNA包括SEQ ID NO.7所示序列，所述3’靶位点的sgRNA序列包括SEQ ID NO.16所示序列。
进一步，所述pCS-4G序列包括SEQ ID NO.27和SEQ ID NO.28所示序列。
进一步，所述打靶载体的基因序列包括SEQ ID NO.26所示序列。
进一步，对F0代小鼠基因型进行鉴定和测序的引物序列包括SEQ ID NO.17～20所示序列。
进一步，对F1代小鼠基因型进行鉴定和测序的引物序列包括SEQ ID NO.21～24所示序列。
本发明的有益效果：
本发明提供了一种能够模拟FOXG1综合征中Foxg1 p.Tyr400X患者的无义突变及其表型的Foxg1 p.Tyr392X小鼠，为神经发育障碍的发病机制研究提供了合适的工具，并为临床开发和干预FOXG1综合征的药物提供了新的思路。对于个性化探讨FOXG1综合征的致病机理、治疗方法、药物筛选等方面有重要意义。此外，Foxg1 p.Tyr392X点突变小鼠模型有利于对Foxg1第392位氨基酸位点、JBD(Foxg1的JARID1B结合结构域)及C端结构域的功能进行更深入的探索，填补该研究方向的空白。
附图说明
图1为本发明的Foxg1 p.Tyr392X点突变小鼠模型的设计方案示意图。
图2为本发明所采用的Southern blot筛选策略示意图。
图3为本发明已插入SEQ ID NO.7所示序列的pCS-4G载体图谱。
图4为本发明已插入SEQ ID NO.16所示序列的pCS-4G载体图谱。
图5为本发明构建的sgRNA的活性检测结果。
图6为本发明构建的打靶载体图谱。
图7为本发明的F0代小鼠PCR鉴定图。
图8为本发明的F1代小鼠PCR鉴定图。
图9为本发明的F1代PCR阳性小鼠的Southern blot检测结果图。
图10为本发明的F1代小鼠基因测序图(c.1176C>A,p.Tyr392*)。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清晰、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创新性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明中涉及的专业术语解释如下表1所示：
表1
实施例1：
本实施例公开了一种Foxg1 p.Tyr392X点突变小鼠模型的构建方法及其应用，包括以下步骤：
(1)打靶载体的构建：
利用CRISPR/Cas9技术，构建针对目的基因的sgRNA，体外转录为mRNA，指导Cas9蛋白在特定位点剪切DNA双链。首先分析FOXG1综合征的病例突变位点和临床症状，该临床病例在FOXG1第400位氨基酸对应的三联密码子由TAC变成TAA(FOXG1编码序列第1176位碱基C突变为碱基A)后，编码酪氨酸的三联密码子变为终止密码子，其症状表现为智力障碍、运动障碍、眼神接触障碍、手部刻板动作、脊柱侧弯、肌张力不足等。接下来根据物种、基因名称知道基因的编码区，分析与人相应的基因组结构，确定在小鼠Foxg1基因的第392位氨基酸引入终止密码子。sgRNA设计在IntronⅠ和3’UTR下游的非保守序列中。5’端和3’端的同源臂分别为1.3kb和1.3kb，设计如图1所示。为了筛选到发生正确重组的基因打靶小鼠，采用PCR和Southern blot方法验证，并同时利用3’Probe(3’端的基因探针)和LR Probe(5’端的基因探针)验证F1代阳性小鼠，具体设计如图2所示。
(2)Cas9/sgRNA设计与构建
基于sgRNA的设计原理，在5’端和3’端靶点区域分别设计了SEQ ID NO.1～8和SEQID NO.9～16所示的sgRNA基因序列。按照设计的sgRNA序列合成Oligos，通过Gibson方法连入pCS-4G载体，载体图谱如图3所示。待连接产物测序正确之后，使用通用扩增引物进行扩增T7启动子及sgRNA核苷酸序列，最后以该PCR产物为模板进行体外转录，得到sgRNA7和sgRNA16的显微注射RNA；并且其制备时反应条件为65℃下反应5min。
之后利用UCATM方式对sgRNA的活性进行检测，活性检测结果如图4所示，最终选择5’端靶点设计的SEQ ID NO.7和3’端靶点设计的SEQ ID NO.16两条sgRNA进行下一步的实验，打靶质粒如图5所示。
(3)胚胎供体小鼠(C57BL/6)超排卵
PMSG(孕马血清促性腺激素)处理供体雌性小鼠，48小时后注射hCG(人绒毛膜促性腺激素)，与雄性小鼠合笼交配，次日取受精卵进行显微注射。
(4)显微注射
将Cas9、sgRNA的显微注射RNA及打靶载体显微注射到小鼠受精卵中，获得F0代小鼠。
(5)F0代小鼠基因型鉴定
由于胚胎早期卵裂速度很快，因此得到的F0代小鼠为嵌合体。设计SEQ ID NO.17～20所示序列的引物，对F0代小鼠鼠尾进行PCR鉴定，得到的F0代小鼠基因型仅供参考，鉴定结果如图6所示。但这次获得的小鼠不能代表其一定为可遗传的基因突变型，可遗传的基因型需待F1代小鼠基因型鉴定后确定。
(6)F1代小鼠基因型及Southern blot鉴定
选择上述F0代小鼠与野生型小鼠交配，生出的小鼠为F1代小鼠，设计SEQ IDNO.21～24所示序列的引物，将F1代PCR鉴定为阳性小鼠(发生基因突变的小鼠)的DNA进行Southern blot检测及测序，结果如7、8、9所示，确认突变型等位基因正确重组，且没有随机插入，代表Foxg1 p.Tyr392X点突变小鼠模型构建成功。
(7)Foxg1 p.Tyr392X点突变小鼠表现状况
对Foxg1 p.Tyr392X点突变小鼠在成年阶段进行观察，发现该小鼠具备智力障碍、运动障碍、重复刻板动作、社交障碍、肌张力不足等特征。
通过本发明得到的Foxg1 p.Tyr392X点突变小鼠模型，能够模拟FOXG1综合征的发病机制，为精准针对FOXG1 p.Tyr400X突变患者开发治疗药物、深入探讨FOXG1综合征的致病机理以及探索基因治疗的方法和可行性，提供了良好的可视化的动物模型。
以上所述的实施例对本发明的技术方案和有益效果进行了详细说明，应理解的是以上所述仅为本发明的具体实施例，并不用于限制本发明。凡在本发明的原则范围内所做的任何修改、补充和等同替换等，均应包含在本发明的保护范围之内。
Claims (10)
1.一种Foxg1 p.Tyr392X点突变小鼠模型，其特征在于，所述小鼠的Foxg1基因编码序列第1176位碱基C突变为碱基A。
2.一种Foxg1 p.Tyr392X点突变小鼠模型的构建方法，其特征在于，包括以下步骤：
根据小鼠Foxg1未突变前基因序列，设计sgRNA序列；
按照所述设计的sgRNA序列合成oligos，所述oligos通过Gibson Assembly方法连入包含Cas9的pCS-4G载体；
设计引物构建打靶载体；
将Cas9、sgRNA和打靶载体显微注射到小鼠受精卵中，得到F0代小鼠，设计引物对F0代小鼠进行测序；
所述F0代小鼠与野生型小鼠交配，得到F1代小鼠，设计引物对F1代小鼠进行测序。
3.根据权利要求2所述的构建方法，其特征在于，所述小鼠Foxg1未突变前基因序列包括SEQ ID NO.25所示序列。
4.根据权利要求2所述的构建方法，其特征在于，所述sgRNA包括在5’靶位点和3’端靶位点分别设计的序列，所述5’靶位点的sgRNA序列包括SEQ ID NO.1～8所示的任一序列，所述3’靶位点的sgRNA序列包括SEQ ID NO.9～16所示的任一序列。
5.根据权利要求2所述的构建方法，其特征在于，所述pCS-4G载体的序列包括SEQ IDNO.27和SEQ ID NO.28所示序列。
6.根据权利要求2所述的构建方法，其特征在于，对所述FO代小鼠基因序列进行鉴定的引物序列包括SEQ ID NO.17～20所示序列。
7.根据权利要求2所述的构建方法，其特征在于，对所述F1代小鼠基因型鉴定和测序的引物序列包括SEQ ID NO.21～24所示序列。
8.根据权利要求2所述的构建方法，其特征在于，所述打靶载体的序列包括SEQ IDNO.26所示序列。
9.通过权利要求2～8任一所述方法获得的Foxg1基因点突变小鼠模型。
10.权利要求1或9所述的Foxg1基因点突变小鼠模型在研究FOXG1综合征领域的应用。
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