CN113005147A - 一种usp8突变的小鼠动物模型的构建方法及其应用 - Google Patents
一种usp8突变的小鼠动物模型的构建方法及其应用 Download PDFInfo
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Abstract
本发明提供一种USP8突变的ACTH腺瘤小鼠动物模型的构建方法及其应用，方法包括：根据基因序列设计sgRNA，构建基因打靶载体peSpCas9‑sgRNA并筛选出活性较高的sgRNA；设计含T7启动子的引物分别扩增eSpCas9及前述活性较高的sgRNA并回收纯化，体外转录试剂盒将eSpCas9、sgRNA分别转录成mRNA；同时，根据靶位点确定供体序列，体外合成Donor DNA oligos；将上述试剂共注射至小鼠受精卵，移植假孕鼠后，获得F0代小鼠；F0小鼠离乳后1周左右剪鼠尾，试剂盒提取基因组DNA，设计特异性引物PCR扩增靶位点附近DNA片段，PCR产物送样测序，获得阳性小鼠。
Description
技术领域
本发明涉及动物模型的构建方法领域，特别涉及一种USP8突变的小鼠动物模型的构建方法及其应用。
背景技术
泛素化特异性蛋白酶USP8突变能诱发ACTH腺瘤。泛素-蛋白酶体系统是细胞内非常重要的蛋白质降解调节系统。近年来，研究发现全基因组外显子测序显示ACTH腺瘤中存在USP8基因体细胞突变，USP8基因突变可能在大量病人中诱发ACTH腺瘤。研究证实全基因组外显子测序显示10例ACTH腺瘤患者中有4例存在USP8基因体细胞突变，所有突变集中在713和720氨基酸之间(USP8 14-3-3结合域内或邻近位置)。USP8包含一个假定的14-3-3结合结构域RSXSXP，由人类USP814外显子的一部分所编码，其在不同的物种中非常保守。USP814-3-3结合域第4个丝氨酸(S718)的磷酸化导致其与14-3-3蛋白的结合和催化功能失活。研究表明，14-3-3结合域氨基酸偏好在每个位置非常相似，第四个位置丝氨酸的磷酸化对其结合能力至关重要。此外，此丝氨酸(S718)的改变(删除或错义突变)在USP8突变的病人中是最常见的(37/75)。
USP8基因突变可能是ACTH垂体腺瘤发病机制的关键事件，因此，需要建立真实模拟USP8突变ACTH腺瘤的人类细胞或动物模型。
发明内容
本发明的第一个目的是提出一种USP8突变的小鼠动物模型的构建方法，以研究ACTH腺瘤的发生机制。
本发明的第二个目的是提出上述方法构建的小鼠模型在ACTH腺瘤研究中的应用。
本发明的第三个目的是得到上述方法获得的小鼠动物模型的USP8突变的细胞。
为解决上述技术问题，本发明通过以下技术方案予以实现：
一种USP8突变的小鼠动物模型的构建方法，基于CRISPR/Cas9基因敲除技术建立USP8 14-3-3BM KO小鼠模型，所述方法包括如下步骤：
步骤一、根据基因序列设计sgRNA，构建基因打靶载体peSpCas9-sgRNA，并筛选出活性较高的sgRNA；
步骤二、设计含T7启动子的引物分别扩增eSpCas9及前述活性较高的sgRNA并回收纯化，体外转录试剂盒将eSpCas9、sgRNA分别转录成mRNA；同时，根据靶位点确定供体序列，体外合成Donor DNA oligos；
步骤三、将上述试剂共注射至小鼠受精卵，移植假孕鼠后，获得F0代小鼠；
步骤四、F0小鼠离乳后1周左右剪鼠尾，试剂盒提取基因组DNA，设计特异性引物PCR扩增靶位点附近DNA片段，PCR产物送样测序，获得阳性小鼠。
作为优选地，本发明中设计的sgRNA序列有SEQ ID NO：1-3。
sgRNA1：GGCCTGAGTGATATCTGGTGAGG(SEQ ID NO：1)；
sgRNA2：TAGGAGCGCTTCAGTTTCGATGG(SEQ ID NO：2)；
sgRNA3：CTCCTCACCAGATATCACTCAGG(SEQ ID NO：3)；
其中sgRNA2为最优sgRNA序列。
作为优选地，构建基因打靶载体peSpCas9-sgRNA的步骤：
Ensembl数据库查询Human USP8基因组DNA序列，根据DNA序列定位至第14个外显子14-3-3蛋白结合结构域，确定其为基因敲除靶位点，设计sgRNA，根据sgRNA设计合成序列互补的DNA oligos，DNA oligos退火并连接至酶切回收的peSpCas9质粒构建打靶载体peSpCas9-sgRNA。
作为优选地，所述含T7启动子的引物序列为TAATACGACTCACTATAGGGAGA(SEQ IDNO：4)。
作为优选地，所述特异性引物的序列为：
Forward Primer:GTCTGTGCTTAGCAAATTCAAGGCC(SEQ ID NO：5)；
ReversePrimer:GGGCATGGTACTGGGAAAGTGCT(SEQ ID NO：6)。
本发明使用CRISPR/Cas9基因编辑技术，通过对小鼠胚胎基因组进行编辑，实现了对USP8特定位点的敲除突变，移植至代孕鼠中繁育出F0代小鼠。剪鼠尾对其进行测序分析是判断是否建模成功的第一步。测序结果提示，本发明构建成功了杂合子和纯合子小鼠，其USP8特定部位均发生不同程度的突变。
附图说明
图1为本发明的构建方法的原理图；
图2为对照组和突变组的垂体组织HE染色对比照片，其中a、b为对照组垂体组织HE染色照片，c、d为突变组的垂体组织HE染色照片；
图3为对照组和突变组的肾上腺组织HE染色对比照片，其中a、b为对照组肾上腺组织HE染色照片，c、d为突变组的肾上腺组织HE染色照片；
图4为对照组和突变组的肾脏组织HE染色对比照片，其中a、b为对照组肾脏组织HE染色照片，c、d为突变组的肾脏组织HE染色照片；
图5为对照组和突变组的肝脏组织HE染色对比照片，其中a、b为对照组肝脏组织HE染色照片，c、d为突变组的肝脏组织HE染色照片；
图6为对照组和突变组的股骨组织HE染色对比照片，其中a、b为对照组股骨组织HE染色照片，c、d为突变组的股骨组织HE染色照片；
图7为对照组和突变组的胫骨组织HE染色对比照片，其中a、b为对照组胫骨组织HE染色照片，c、d为突变组的胫骨组织HE染色照片；
图8为对照组和模型组的颅脑7.0T磁共振扫描图，其中a、b为颅脑7.0T磁共振扫描图，c、d为突变组的颅脑7.0T磁共振扫描图；白色箭头所示为小鼠垂体；
图9为对照组和模型组的血液皮质醇浓度检测图；
图10为对照组和模型组24小时尿皮质醇检测图；
图11为对照组和模型组的血液ACTH检测图；
图12为对照组小鼠心脏B超检查图，其中，a、b为对照组1的小鼠心脏B超检查图；c、d为对照组2的小鼠心脏B超检查图；e、f为对照组3的小鼠心脏B超检查图；
图13为模型组的小鼠心脏B超检查图；
图14为阳性小鼠垂体HE染色图，其中a、b为对照组垂体组织HE染色照片，c、d为突变组的垂体组织HE染色照片；
图15为对照组和突变组的垂体组织网状纤维染色对比图片，其中a为对照组垂体网状纤维染色，b为突变组垂体网状纤维染色。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚，下面将结合具体实施例进行详细描述。
一种USP8突变的小鼠动物模型的构建方法，基于CRISPR/Cas9基因敲除技术建立USP8 14-3-3BM KO小鼠模型，其特征在于，所述方法包括如下步骤：
步骤一、根据基因序列设计sgRNA，构建基因打靶载体peSpCas9-sgRNA并筛选出活性较高的sgRNA；
设计的sgRNA有sgRNA1-3。
sgRNA1：GGCCTGAGTGATATCTGGTGAGG；
sgRNA2：TAGGAGCGCTTCAGTTTCGATGG；
sgRNA3：CTCCTCACCAGATATCACTCAGG；
活性较高的sgRNA为sgRNA2。
步骤二、设计含T7启动子的引物分别扩增eSpCas9及前述活性较高的sgRNA并回收纯化，体外转录试剂盒将eSpCas9、sgRNA分别转录成mRNA；同时，根据靶位点确定供体序列，体外合成Donor DNA oligos；
步骤三、将上述试剂共注射至小鼠受精卵，移植假孕鼠后，获得F0代小鼠；
步骤四、F0小鼠离乳后1周左右剪鼠尾，试剂盒提取基因组DNA，设计特异性引物PCR扩增靶位点附近DNA片段，PCR产物送样测序，获得阳性小鼠。
本发明的构建方法的原理图如图1所示，其中(-)表示缺失序列。
对获得的USP8突变小鼠的一些特性进行了分析，挖掘该模型能否诱发ACTH腺瘤。特性分析的过程：
(1)小动物核磁共振仪观测小鼠垂体成瘤情况：利用中山大学中山医学院小动物分子影像平台完成对小鼠进行磁共振检测，观察肿瘤形成。
(2)各个器官组织HE染色：肝脏、肾脏等器官观察USP8突变ACTH小鼠是否引起其他器官的损伤；同时，利用HE染色观察垂体腺组织是否存在肿瘤性改变，肾上腺是否出现病理性改变及骨组织是否出现骨质疏松等。通过垂体腺组织的网状纤维染色，观察突变小鼠组织中的网状纤维是否被破坏，间接诊断是否存在肿瘤性改变。
(3)血液生化及激素分析：血浆样品取样参考文献进行，每个血清/血浆体积需要500μL左右，于800g 4℃下进行离心10min进行分离，并于-20℃保存备用。使用时进行血皮质醇等指标分析；为检测皮质醇的昼夜节律，于昼夜的谷值(早上，开灯后1小时)及昼夜峰值(傍晚，关灯前1小时)收集血液样品，使用ELISA试剂盒测量血浆皮质醇浓度及ACTH激素。
(4)小鼠尿液生化分析：小鼠单独饲养在代谢笼里24小时。记录体重、24小时水及食物消耗及24小时尿量。分析前，所有摄入食物或水及尿量的测量用每100g体重来表达。用来分析的尿液样品在800×g，4℃下离心10分钟，储存在-20℃备用。稀释(按1:4蒸馏水稀释)及未稀释样品测定尿皮质醇。
(5)小鼠心血管功能检测：利用小动物血管多谱勒测量小鼠心脏超声，与野生型小鼠对比，观察心脏结构的变化。
为了了解USP8突变后是否影响小鼠的器官功能，实施例做了垂体、肝脏、肾脏、肾上腺、股骨、胫骨等组织切片并进行HE染色，与对照组小鼠的对比结果如图2-7所示，提示基因编辑小鼠并未影响其他器官组织的发育状态。
颅脑7.0T磁共振扫描显示如图8所示，USP8 KO小鼠的垂体中存在部分信号不均的现象，而对照组则极少存在该情况，然而，小鼠脑垂体较小，加上ACTH腺瘤以微腺瘤居多，部分不均匀的信号影无法很好的下准确的临床诊断。因此，继续对血液和尿液的相关激素进行检测。尿液皮质醇浓度检测结果如图9所示，血液皮质醇浓度检测结果如图10所示，提示基因编辑小鼠的体液皮质醇浓度比对照小鼠的高，差异具有统计学意义。值得注意的是，血液ACTH检测如图11所示，未见基因编辑小鼠与野生型小鼠的差异。造成该结果的可能原因是皮质醇和ACTH受动物情绪、健康情况、饲养情况等影响，容易造成短暂的分泌紊乱，从而产生假阴性或者假阳性结果。ACTH腺瘤能明显影响心脏功能。因此对小鼠的心脏进行了超声检查，探索其收缩功能是否发生改变。结果如图12和13所示，对USP8KO和野生型小鼠的心脏体积、左心室射血分数、心室壁厚度等数据进行了检查，发现突变小鼠的心功能对比大部分的野生小鼠均发生了改变。小鼠心功能检测数据如表1所示。
表1
| Index | EF(％) | FS(％) | LV Mass(mg) | LV Vol；d(uL) | LV Vol；s(uL) |
| WT1 | 87.8 | 56.6 | 67.75617 | 43.789127 | 5.33633 |
| WT2 | 92.3 | 61.9 | 91.035512 | 12.667354 | 0.972253 |
| WT3 | 93.2 | 64.3 | 63.717888 | 24.81333 | 1.691301 |
| MUT1 | 88.7 | 57.5 | 51.524739 | 36.145594 | 4.097926 |
最后，将阳性鼠进行了解剖，取下垂体组织进行HE染色及及网状纤维染色，并与对比例进行对照。染色结果如图14、15所示，突变组小鼠垂体组织细胞排列紊乱，网状纤维不完整，符合肿瘤性改变。考虑ACTH腺瘤病程较长，因此尚未出现明显的影像学及血液学改变。后续将继续观察模型的相关疾病指标的改变。
以上所述是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明所述原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中山大学附属第一医院
<120> 一种USP8突变的小鼠动物模型的构建方法及其应用
<141> 2021-03-01
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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<212> DNA
<213> 人工序列(Artificial Sequence)
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Claims (6)
1.一种USP8突变的小鼠动物模型的构建方法，基于CRISPR/Cas9基因敲除技术建立USP8 14-3-3 BM KO小鼠模型，其特征在于，所述方法包括如下步骤：
步骤一、设计sgRNA1-3，构建基因打靶载体peSpCas9-sgRNA并筛选出活性较高的sgRNA；
其中，sgRNA1的序列为SEQ ID NO：1所示，sgRNA2的序列为SEQ ID NO：2所示，sgRNA3的序列为SEQ ID NO：3所示；
活性较高的sgRNA为sgRNA2；
步骤二、设计含T7启动子的引物分别扩增eSpCas9及前述活性较高的sgRNA并回收纯化，体外转录试剂盒将eSpCas9、sgRNA分别转录成mRNA；同时，根据靶位点确定供体序列，体外合成Donor DNA oligos；含T7启动子的引物序列如SEQ ID NO：4所示；
步骤三、将上述试剂共注射至小鼠受精卵，移植假孕鼠后，获得F0代小鼠即为阳性小鼠。
2.根据权利要求1所述的一种USP8突变的小鼠动物模型的构建方法，其特征在于，还包括步骤四：F0小鼠离乳后1周左右剪鼠尾，试剂盒提取基因组DNA，设计特异性引物PCR扩增靶位点附近DNA片段，PCR产物送样测序，检验是否为阳性小鼠，其中，特异性引物序列如SEQID NO：5～6所示。
3.根据权利要求1或2所述的一种USP8突变的小鼠动物模型的构建方法在ACTH腺瘤研究中的应用。
4.根据权利要求1或2所述的方法获得的小鼠动物模型的USP8突变的细胞。
5.一种基于CRISPR/Cas9基因敲除技术建立USP8 14-3-3 BM KO小鼠模型的sgRNA，其特征在于，包括sgRNA1-3，所述sgRNA1如SEQ ID NO:1所示，所述sgRNA2如SEQ ID NO:2所示，所述sgRNA3如SEQ ID NO:3所示。
6.根据权利要求5所述的sgRNA在建立基因缺陷小鼠中的应用。
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