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CN108148837A - ApoE-CRISPR/Cas9载体及其在敲除ApoE基因中的应用 - Google Patents

ApoE-CRISPR/Cas9载体及其在敲除ApoE基因中的应用 Download PDF

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CN108148837A
CN108148837A CN201810030989.9A CN201810030989A CN108148837A CN 108148837 A CN108148837 A CN 108148837A CN 201810030989 A CN201810030989 A CN 201810030989A CN 108148837 A CN108148837 A CN 108148837A
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apoe
crispr
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sgrna
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戴凡
戴一凡
杨海元
王盈
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Nanjing Medical University
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Abstract

本发明公开了特异性靶向ApoE基因第二外显子的SgRNA，所述SgRNA的核苷酸序列为GCTTCTGGGATTACCTGCGC。本发明还公开了含所述SgRNA的ApoE‑CRISPR/Cas9载体，ApoE‑CRISPR/Cas9载体在构建ApoE基因敲除的哺乳动物模型中的应用及在制备ApoE基因敲除试剂盒中的应用。本发明基于CRISPR/Cas9技术构建了ApoE基因敲除的哺乳动物模型，敲除ApoE基因能影响哺乳动物体内的脂质代谢，加速动脉粥样硬化等脂代谢相关疾病的发病进程，从而加速哺乳动物相应疾病模型的造模进程，为动脉粥样硬化等疾病的研究提供动物实验依据。

Description

ApoE-CRISPR/Cas9载体及其在敲除ApoE基因中的应用
技术领域
本发明属于基因工程技术领域，涉及基因敲除，具体涉及ApoE-CRISPR/Cas9载体及其在敲除ApoE基因中的应用。
背景技术
心血管疾病是我国居民健康的“头号杀手”，《中国心血管病报告2015》报告显示：我国每5例死亡病例中，有2例死于心血管病。动脉粥样硬化(atherosclerosis,AS)是动脉管壁发生的一种慢性炎症性疾病，是心脑血管疾病的主要病理基础。AS表现为大中型弹性血管，肌性动脉壁内膜及内膜下出现脂质沉积，形成粥样病灶或纤维脂质斑块，造成动脉管腔狭窄，随后斑块破裂脱落，在狭窄的动脉内形成血栓，导致缺血性心脑血管疾病和死亡的发生。另外，粥样病灶也可以破坏血管平滑肌，导致粥样血管瘤(atheroscleroticaneurysms)的形成，加上其它因素，如高血压等，常常引起腹部，胸部和脑部血管的破裂，致命或致残。动脉粥样硬化不稳定斑块(易损斑块)发生溃疡、破裂伴血栓形成，是导致临床急性事件，如不稳定型心绞痛、急性心肌梗死、卒中和猝死的主要原因。在大多数西方国家，AS是导致人死亡的首位原因。而在我国，随着经济的发展和人民生活水平的不断提高，AS的发病率、死亡率也有不断加剧的趋势，严重威胁着人民的生存质量与生命健康。由于AS的高发病率和致死率，其发病机制和防治策略一直是国内外医学研究的热点。
载脂蛋白E(apolipoprotein，ApoE)是血浆极低密度脂蛋白(VLDL)、高密度脂蛋白(HDL)、乳糜微粒(CM)等脂蛋白的组分，可作为配体结合相应的脂蛋白受体或相关蛋白来介导血浆脂蛋白的清除和血浆胆固醇水平的调节。人ApoE由29个氨基酸组成，首先在肝脏内合成，在脑、肾脏和脾脏等其他组织中也有表达。ApoE基因位点具有多态性，主要有3个等位基因：ε2、ε3、ε4，分别编码对应的3种蛋白ApoE2、ApoE3、ApoE4。ApoE多态性是AS早期及发展过程中个体差异的重要原因，ApoE4/4异构体可显著地升高人的总胆固醇和三酰甘油的水平并使之易患AS，人ApoE基因缺失则易形成高脂血症病和AS。ApoE基因敲除(ApoE KO)小鼠的血浆胆固醇显著升高，表现为脂蛋白清除障碍，在短时间内即可形成复杂的AS病变。而在ApoE基因敲除小鼠的肝组织中用腺病毒载体表达人的ApoE蛋白、在肌肉中注射人的ApoEcDNA都能抑制其AS的发生。以上研究表明ApoE是AS发生发展中的一个重要分子标靶。
人类疾病动物模型是基础研究向临床转化的重要桥梁。一个好的疾病动物模型应具有与人类疾病发病机制同源性、表型特征一致性的特点，而且易创建、重现性好、经济。缺乏合适的动物模型是许多重大疾病发病机制分析和药物筛选、评价的主要瓶颈。因此，深入研究和发现易损斑块的致病机理、鉴定可预测心血管事件的可靠生物标记物，发现新的干预靶点、寻找有效的治疗方法，是我国当前迫切需要解决的重大社会和医学问题。不断开发、建立与完善各类疾病动物模型是转化医学研究的一个重要内容。
发明内容
发明目的：本发明的目的是提供ApoE-CRISPR/Cas9载体；进一步的目的是提供ApoE-CRISPR/Cas9载体在构建ApoE基因敲除的哺乳动物模型中的应用；更进一步的目的是提供ApoE-CRISPR/Cas9载体在制备ApoE基因敲除试剂盒中的应用。
技术方案：本发明所述特异性靶向ApoE基因第二外显子的SgRNA，该位点适合靶点设计原则，所述SgRNA的核苷酸序列如SEQ ID No:1所示。
进一步地，本发明提供含有所述SgRNA的ApoE-CRISPR/Cas9载体，将SgRNA和Cas9构建在一个质粒载体上，细胞转染效率会更高，更容易筛选阳性克隆。
优选地，所述ApoE-CRISPR/Cas9载体的核苷酸序列如SEQ ID No:2所示。所述ApoE-CRISPR/Cas9载体包括U6基因启动子，用于启动SgRNA表达；SgRNA序列；CBh基因启动子，用于启动Cas9编码基因表达；Cas9编码基因。其中第725-965碱基为U6基因启动子区，第974-993碱基为sgRNA，第1166-1964碱基为CBh基因启动子的编码区，第2097-6197碱基为Cas9基因的编码区。
所述ApoE-CRISPR/Cas9载体按如下方法制备得到：
1)根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，在ApoE基因上寻找靶点位置；
2)购买表达hSpCas9和gRNA的pX330骨架质粒(Addgene plasmid 423230)；
3)公司合成5’端磷酸化寡核苷酸链SgRNA序列GCTTCTGGGATTACCTGCGC，将SgRNA序列克隆到pX330骨架载体上，具体步骤如下：
①用限制性内切酶BbsI消化1ug pX330质粒；
②酶切的pX330质粒跑琼脂糖胶分离，用胶回收试剂盒纯化回收酶切产物；
③公司合成的5’端磷酸化寡核苷酸链SgRNA序列按照以下程序退火：
37℃30min
95℃5min然后以5℃/min的速率降至25℃。
④按照以下体系启动连接反应：室温反应10min
⑤用质粒安全核酸外切酶处理连接体系，去除错误链接质粒：
37℃反应30min
⑥转化
1)取50μL感受态细胞(TIANGEN)置于冰浴中；
2)向装有感受态细胞的离心管中加入15μL步骤⑤得到的去错误连接质粒溶液，混匀后在冰浴中静置30min；
3)将冰浴30min的感受态细胞置于42℃水浴中60～90s，然后迅速转移到冰浴中，使细胞冷却2～3min；
4)向离心管中加入900μL无菌的LB培养基(不含抗生素)，混匀后置于37℃摇床150rpm振荡培养45min；
5)将离心管放到离心机中12000rpm离心5min，然后弃去900μL上清，用剩余的100μL上清重悬感受态细胞沉淀，然后将重悬的感受态细胞加到含相应抗生素的LB固体琼脂培养基上，用无菌的涂布棒将感受态细胞涂布均匀；将涂布有感受态细胞的LB固体琼脂培养基倒置于37℃培养箱中培养12～16h。
⑦小提质粒，从公司测序，鉴定打靶质粒构建成功。
更进一步地，本发明提供RISPR/Cas9载体在构建ApoE基因敲除的哺乳动物模型中的应用，包括如下步骤：
(1)将ApoE-CRISPR/Cas9载体转化至哺乳动物的胎儿成纤维细胞中；
(2)使用G418抗生素对步骤(1)得到的成纤维细胞进行抗性筛选，将具有抗性的成纤维细胞进行基因测序，用于确定ApoE基因序列发生了改变，保留成功转基因的成纤维细胞；
(3)将步骤(2)得到的成纤维细胞的细胞核移植到去核的哺乳动物卵母细胞中培养至囊胚阶段；
(4)将步骤(3)得到的囊胚移植到代孕哺乳动物中进行饲养，生产；
(5)提取步骤(4)生产的哺乳动物的基因组，利用PCR引物进行扩增，进行基因型鉴定。
其中，步骤(1)中的转化方法为核转染；步骤(3)中的核移植方法为体细胞克隆技术；步骤(4)中的囊胚移植方法为胚胎移植技术。步骤(1)～(5)中所述哺乳动物为巴马小型猪。
步骤(5)中所述PCR引物为：正向引物为5’-gcagggcgtgagcattagat-3’，反向引物为5’-gagttggcgacaaggacagaa-3’。
更进一步地，本发明提供ApoE-CRISPR/Cas9载体在制备ApoE基因敲除试剂盒中的应用。
有益效果：本发明基于CRISPR/Cas9技术构建了ApoE基因敲除的哺乳动物模型，敲除ApoE基因能影响哺乳动物体内的脂质代谢，加速动脉粥样硬化等脂代谢相关疾病的发病进程，从而加速哺乳动物相应疾病模型的造模进程，为动脉粥样硬化等疾病的研究提供动物实验依据。
附图说明
图1为ApoE基因CRISPR/Cas9靶点及引物示意图；
图2为适用于ApoE基因敲除的载体ApoE-PX330的示意图；
图3为ApoE基因敲除型巴马小型猪的蛋白免疫印迹(Western Blotting)结果；
图4为ApoE基因敲除型巴马小型猪的主动脉血管经苏丹Ⅲ染液的结果；
图5为ApoE基因敲除型巴马小型猪的主动脉血管的病理切片图。
具体实施方式
实施例1ApoE-PX330载体的构建
首先根据Genbank中公布的猪ApoE基因序列(SSU70240)，选取ApoE基因的2号外显子exon2作为CRISPR/Cas9靶点，根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，设计向导序列GCTTCTGGGATTACCTGCGC见图1。
ApoE-CRISPR/Cas9载体按如下方法制备得到：
步骤一、根据cas9靶点设计原则：5’端为G，3’端为PAM序列(NGG)，在ApoE基因上寻找靶点位置；
步骤二、购买表达hSpCas9和gRNA的pX330骨架质粒(Addgene plasmid 423230)；
步骤三、公司合成5’端磷酸化寡核苷酸链SgRNA序列GCTTCTGGGATTACCTGCGC。
将SgRNA序列克隆到pX330骨架载体上，具体步骤如下：
1、用限制性内切酶BbsI消化1ug pX330质粒；
2、酶切的pX330质粒跑琼脂糖凝胶(琼脂糖凝胶浓度1％，即1g琼脂糖凝胶加入到100mL电泳缓冲液中)分离，用胶回收试剂盒(QIAGEN)纯化回收酶切产物；
3、公司合成的5’端磷酸化寡核苷酸链SgRNA序列按照以下程序退火：
37℃30min
95℃5min然后以5℃/min的速率降至25℃。
4、按照以下体系启动连接反应：室温反应10min
5、用质粒安全核酸外切酶处理连接体系，去除错误链接质粒：
37℃反应30min
6、转化
(1)取50μL感受态细胞(TIANGEN)置于冰浴中；
(2)向装有感受态细胞的离心管中加入15μL步骤5得到的去错误连接质粒溶液，混匀后在冰浴中静置30min；
(3)将冰浴30min的感受态细胞置于42℃水浴中60～90s，然后迅速转移到冰浴中，使细胞冷却2～3min；
(4)向离心管中加入900μL无菌的LB培养基(不含抗生素)，混匀后置于37℃摇床150rpm振荡培养45min；
(5)将离心管放到离心机中12000rpm离心5min，然后弃去900μL上清，用剩余的100μL上清重悬感受态细胞沉淀，然后将重悬的感受态细胞加到含相应抗生素的LB固体琼脂培养基上，用无菌的涂布棒将感受态细胞涂布均匀；将涂布有感受态细胞的LB固体琼脂培养基倒置于37℃培养箱中培养12～16h。
7、小提质粒，从公司测序，鉴定打靶质粒构建成功。
所构建的CRSAPR/Cas9载体命名为ApoE-PX330，全核苷酸序列如SEQ ID No:2所示，总长8508bp。这种能高效在巴马小型猪体内敲除ApoE基因的质粒载体包含U6基因启动子、靶向猪ApoE的向导序列ApoE/sgRNA-X，CBh基因启动子，Cas9编码基因，见图2，其中725-965bp为U6基因启动子区，第974-993bp为ApoE/sgRNA-X，第1166-1964bp为CBh基因启动子的编码区，第2097-6197bp为Cas9基因的编码区。能广泛性表达的U6基因的启动子可保证下游基因广泛性表达。
实施例2利用体细胞克隆的方法创制ApoE基因敲除的巴马小型猪细胞
步骤一、猪原代成纤维细胞复苏
1、从液氮中取出冻存的原代猪成纤维细胞，在37℃水浴中解冻；
2、将解冻的细胞转入无菌的15mL离心管中，然后加入3mL细胞培养基，1500rpm离心5min；
其中，细胞完全培养基的配方为：16％胎牛血清(Gibco)+84％DMEM培养基(Gibco)，16％和84％为体积百分比。
3、弃去上清，加入2mL完全培养基重悬细胞沉淀，然后将重悬的细胞铺入6cm细胞培养皿中，补加2mL完全培养基，置于37℃，5％CO2(体积百分比)的恒温培养箱中进行培养；
4、将细胞培养至长满皿底90％左右时使用0.05％(5g/100mL)的胰蛋白酶将细胞消化下来，然后加入完全培养基终止消化，将细胞悬液转入15mL离心管中，1500rpm离心5min，弃去上清，使用2mL完全培养基重悬细胞，对细胞计数，将细胞总量调整至1.5×106以备下一步核转染实验。
步骤二、使用构建好的ApoE-PX330和tdTomato质粒(Clontech,PT4069-5)共转染猪原代成纤维细胞
使用哺乳动物成纤维细胞核转染试剂盒(Lonza)与Lonza NucleofactorTM2b核转仪进行核转染实验
1、配制核转染反应液，体系如下：
核转染基本溶液 82μL
补充成分 8μL
2、将构建好的ApoE-PX330质粒与Tdtomato质粒按照质量比5:1的比例加入本步骤1获得的100μL核转反应液中混匀，过程中注意切勿产生气泡；
3、将步骤一制备得到的细胞悬液使用DPBS杜氏磷酸缓冲液(Gibco)洗一遍，1500rpm离心5min，弃去上清，使用本步骤2中含有质粒的核转反应液重悬细胞，重悬过程中要避免气泡的产生；
4、将该核转体系小心加入到试剂盒带有的电转杯中，注意防止气泡。电转杯放置于Lonza核转仪的杯槽内，选择U023核转程序，电击转染后立即在超净台内将电转杯中液体轻柔吸出，转入到2mL含体积百分比为16％胎牛血清的DMEM完全培养基中，轻轻混匀；
5、准备含8mL完全培养基的培养皿(10cm)若干，吸取核转后的细胞悬液10μL加入含有完全培养基的培养皿中，混匀，在显微镜下观察细胞数量，调整加入更多细胞悬液，使得培养皿在显微镜下一个视野内约有20～30个细胞，其余皿均按照此细胞悬液最终用量加入，混匀后放置于37℃，5％CO2的恒温培养箱中进行培养。
步骤三、ApoE基因敲除细胞系的筛选
1、将步骤二所得细胞培养24h后将细胞培养基更换为含有1mg/mL G418的完全培养基，放置于37℃，5％CO2的恒温培养箱中进行培养，每2～3天更换一次细胞培养基，期间根据细胞生长状况逐渐降低G418的药物浓度，G418终浓度为0.3mg/mL，培养10～14天左右培养皿中会陆续长出G418抗性的单克隆细胞系；
2、使用克隆环挑取细胞系，将挑取的单克隆细胞系接种于铺有0.3mg/mLG418完全培养基的24孔板中，放置于37℃，5％CO2的恒温培养箱中进行培养，每2～3天换一次细胞培养基；
3、待24孔板的孔中细胞长满孔底，使用胰蛋白酶消化并收集细胞，其中4/5细胞接种到含有0.3mg/mL G418完全培养基的6孔板中，剩余的1/5的细胞留在24孔板中继续培养；
4、待6孔板细胞铺满孔底后使用0.05％(5g/100mL)的胰蛋白酶消化并收集细胞，使用细胞冻存液(90％胎牛血清+10％DMSO，体积比)将细胞冻存；
步骤四、ApoE敲除细胞系的鉴定
1、待24孔板中细胞长满孔底后使用0.05％(5g/100mL)的胰蛋白酶消化并收集细胞，然后在细胞中加入25ml NP-40裂解液裂解细胞提取细胞基因组DNA，裂解程序为：55℃60min——95℃5min——4℃，反应结束后基因组DNA于-20℃保存；
2、针对ApoE基因靶点信息设计相应的PCR引物，PCR引物序列：正向引物为：5’-gcagggcgtgagcattagat-3’，反向引物为：5’-gagttggcgacaaggacagaa-3’，PCR目的产物长度为619bp；
3、使用PCR反应扩增ApoE靶点基因，PCR反应体系如下：
反应条件如下
4、将PCR反应产物进行琼脂糖凝胶电泳(1％，即1g琼脂糖凝胶加入到100mL电泳缓冲液中)，电泳结束后在紫外线下切下大小约为619b p的目的条带，然后使用胶回收试剂盒(QIAGEN)回收目的条带，并使用NanoDrop 200测定回收的PCR产物的浓度；
5、将回收的PCR产物使用TAKARA pMDTM18-T Vector Cloning Kit链接T载体，T载体反应体系如下：
pMD18-T vector 1μL
胶回收PCR产物 81.7ng*
ddH2O 补齐体系至10uL
*注：TAKARA pMDTM18-T Vector Cloning Kit说明书上对Insert DNA(本次为胶回收PCR产物)用量的要求0.1～0.3pM，本次选取0.2pM，用量计算方法为：Insert DNA的使用量(ng)＝nmol数×660×Insert DNA的bp数。
T载体链接的反应条件为16℃反应30min；
6、将本步骤5所得的T载体链接产物使用感受态细胞(TIANGEN)进行转化，转化后将感受态细胞涂布于Amp抗性的LB琼脂固体培养基上，37℃恒温培养箱培养过夜；
从培养过夜的培养基上挑取10～15个单克隆菌落送测序公司进行测序，然后将测序结果与ApoE靶点信息进行比对，从而判断该细胞系是否为ApoE基因敲除细胞系；
本次挑取的单克隆细胞系共28个，其中ApoE基因双敲除细胞系14个。细胞基因型情况见表1：
表1ApoE基因敲除的巴马小型猪成纤维细胞的基因鉴定
步骤五、体细胞核移植
1、从屠宰场购买六月龄以上的母猪卵巢，人工抽取卵泡中未成熟的卵母细胞，在显微镜下挑取质量较好的卵母细胞并置于38.5℃，5％CO2恒温培养箱中培养42～44h至卵母细胞成熟；
2、利用显微操作系统将本步骤(1)中成熟的卵母细胞去核，然后复苏步骤四获得的ApoE敲除单克隆细胞系，将ApoE敲除细胞作为核供体注入去核卵母细胞中，每个去核卵母细胞注射一个ApoE敲除细胞；
3、将注射好的细胞利用电融合技术将核移植后得重构胚胎激活，将胚胎置于38.5℃培养箱培养5天发育成桑椹胚；
4、将发育情况良好的胚胎移植到代孕母猪的子宫中，小心护理代孕母猪，移植一个月后使用B超检测受体猪的怀孕情况，期间及时监控直至代孕母猪分娩。
步骤六、ApoE基因敲除巴马小型猪的基因型分析
1、ApoE基因敲除小猪出生后剪取小猪耳部组织，然后使用血液/细胞/组织基因组DNA提取试剂盒(TIANGEN)提取小猪基因组DNA；
2、使用本步骤1中所得小猪基因组DNA进行PCR反应，PCR反应条件同步骤四3，然后将PCR反应产物送测序公司进行测序，将测序结果与ApoE基因靶点序列进行比对。
PCR扩增所得到的546bp的片段为阳性片断，本次共出生10只巴马小型猪编号为M1～M10，出生小猪的基因型检测结果见表2，其中WT为野生巴马小型猪，M1，M2，M3，M6，M7，M10为ApoE基因敲低型巴马小型猪，M4，M5，M8，M9为ApoE基因敲除型巴马小型猪，其中Δ表示基因敲除。
表2ApoE基因敲除的巴马小型猪的基因鉴定
实施例3ApoE基因敲除巴马小型猪的表型分析。
1、ApoE基因敲除型巴马小型猪体内ApoE蛋白表达情况检测
对断奶后的ApoE基因敲除小型猪使用不抗凝采血管采血，静置1h以上，待管内有血清析出后将采血管放入离心机，3000r/min离心10min后取上层澄清液体即为血清。将血清稀释后进行聚丙烯酰胺凝胶(SDS-PAGE)进行蛋白电泳，然后使用转印装置将电泳好的聚丙烯酰胺凝胶上的蛋白转印到PVDF膜上，最后使用Apolipoprotein E/ApoE antibody(NOVUS NBP1-31123)对ApoE基因敲除情况进行检测，检测结果见图3，其中WT为野生型巴马小型猪，M1，M2，M3，M6，M7，M10为ApoE基因敲低型巴马小型猪，M4，M5，M8，M9为ApoE基因敲除型巴马小型猪。
2、高脂喂养后ApoE基因敲除型巴马小型猪的胸主动脉进行苏丹Ⅲ染色分析脂肪含量
将高脂喂养6个月后的巴马小型猪用呼吸机进行吸入式麻醉，待小猪麻醉后采用心脏灌流的方式对小猪进行处死，然后将小猪的胸主动脉完整取出。将取出的胸主动脉纵切分剖开并放入4g/100ml的多聚甲醛水溶液中固定24h以上。将固定好的胸主动脉用蒸馏水冲洗1min，再使用体积比为70％乙醇水溶液中清洗1mi，然后将组织放入事先配置的苏丹Ⅲ染液中染色30min。将染色完毕的胸主动脉使用体积比为70％乙醇水溶液多次清洗，直至胸主动脉血管壁上无病变组织变成黄白色。染色结果见图4，其中颜色加深的地方(即箭头所指部分)为动脉粥样硬化斑。
3、高脂喂养后ApoE敲除型巴马小型猪胸主动脉病理切片分析。
从固定好的猪胸主动脉组织取下大小合适的组织块，然后将组织块置于石蜡包埋筐中进行脱水，脱水的步骤为：75％乙醇4h，85％乙醇2h,，90％乙醇2h，体积比为95％乙醇水溶液1h，无水乙醇I 30min，无水乙醇II 30min(即无水乙醇脱水2次)。脱水结束后对脱水的组织块进行透明，透明的步骤为：醇苯溶液(乙醇：二甲苯1:1，体积比)10min，二甲苯I10min，二甲苯II 10min。透明结束后将组织块进行浸蜡，浸蜡的步骤为：蜡I 1小时，蜡II 1小时，蜡III 1小时。浸蜡结束后将组织块使用包埋机进行包埋，包埋后切片，切片厚度为5μm。将切好的组织切片进行H/E染色，染色的步骤为：(1)石蜡切片脱蜡至水溶液：依次将切片放入二甲苯I 20min-二甲苯II 20min-无水乙醇I10min-无水乙醇II10min–体积比为95％乙醇水溶液5min–体积比为90％乙醇水溶液5min–体积比为80％乙醇水溶液5min–体积比为70％乙醇水溶液5min-蒸馏水洗。(2)苏木素染细胞核：切片入Harris苏木素染3～8min，自来水洗，1％的盐酸酒精(36％～38％浓盐酸和无水乙醇的体积比)溶液中放置数秒，自来水冲洗，0.6％氨水(25％～28％浓氨水和水的体积比)返蓝，流水冲洗。(3)伊红染细胞质：切片入伊红染液中染色1～3min。(4)脱水封片：将切片依次放入体积比为95％乙醇水溶液I5min-体积比为95％乙醇水溶液II 5min-无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-二甲苯Ⅰ5min-二甲苯Ⅱ5min中脱水透明，将切片从二甲苯拿出来稍晾干，中性树胶封片。封片之后放在显微镜下镜检，镜检结果见图5，其中箭头所指部分为动脉粥样硬化斑。
序列表
<110> 南京医科大学
<120> ApoE-CRISPR/Cas9载体及其在敲除ApoE基因中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gcttctggga ttacctgcgc 20
<210> 2
<211> 8508
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag 60
atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa 120
aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg 180
aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag 240
ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg 300
ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga 360
tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc 420
ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc 480
acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga 540
gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt 600
cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg 660
aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac 720
atgtgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag 780
agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg 840
tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta 900
tcatatgctt accgtaactt gaaagtattt cgatttcttg gctttatata tcttgtggaa 960
aggacgaaac accgcttctg ggattacctg cgcgttttag agctagaaat agcaagttaa 1020
aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct tttttgtttt 1080
agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct 1140
gcagacaaat ggctctagag gtacccgtta cataacttac ggtaaatggc ccgcctggct 1200
gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg actttccatt 1260
gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 1320
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattgtg 1380
cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 1440
ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct 1500
ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg 1560
gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga 1620
ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg 1680
cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg 1740
cgacgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc gccccggctc 1800
tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc tccgggctgt 1860
aattagctga gcaagaggta agggtttaag ggatggttgg ttggtggggt attaatgttt 1920
aattacctgg agcacctgcc tgaaatcact ttttttcagg ttggaccggt gccaccatgg 1980
actataagga ccacgacgga gactacaagg atcatgatat tgattacaaa gacgatgacg 2040
ataagatggc cccaaagaag aagcggaagg tcggtatcca cggagtccca gcagccgaca 2100
agaagtacag catcggcctg gacatcggca ccaactctgt gggctgggcc gtgatcaccg 2160
acgagtacaa ggtgcccagc aagaaattca aggtgctggg caacaccgac cggcacagca 2220
tcaagaagaa cctgatcgga gccctgctgt tcgacagcgg cgaaacagcc gaggccaccc 2280
ggctgaagag aaccgccaga agaagataca ccagacggaa gaaccggatc tgctatctgc 2340
aagagatctt cagcaacgag atggccaagg tggacgacag cttcttccac agactggaag 2400
agtccttcct ggtggaagag gataagaagc acgagcggca ccccatcttc ggcaacatcg 2460
tggacgaggt ggcctaccac gagaagtacc ccaccatcta ccacctgaga aagaaactgg 2520
tggacagcac cgacaaggcc gacctgcggc tgatctatct ggccctggcc cacatgatca 2580
agttccgggg ccacttcctg atcgagggcg acctgaaccc cgacaacagc gacgtggaca 2640
agctgttcat ccagctggtg cagacctaca accagctgtt cgaggaaaac cccatcaacg 2700
ccagcggcgt ggacgccaag gccatcctgt ctgccagact gagcaagagc agacggctgg 2760
aaaatctgat cgcccagctg cccggcgaga agaagaatgg cctgttcgga aacctgattg 2820
ccctgagcct gggcctgacc cccaacttca agagcaactt cgacctggcc gaggatgcca 2880
aactgcagct gagcaaggac acctacgacg acgacctgga caacctgctg gcccagatcg 2940
gcgaccagta cgccgacctg tttctggccg ccaagaacct gtccgacgcc atcctgctga 3000
gcgacatcct gagagtgaac accgagatca ccaaggcccc cctgagcgcc tctatgatca 3060
agagatacga cgagcaccac caggacctga ccctgctgaa agctctcgtg cggcagcagc 3120
tgcctgagaa gtacaaagag attttcttcg accagagcaa gaacggctac gccggctaca 3180
ttgacggcgg agccagccag gaagagttct acaagttcat caagcccatc ctggaaaaga 3240
tggacggcac cgaggaactg ctcgtgaagc tgaacagaga ggacctgctg cggaagcagc 3300
ggaccttcga caacggcagc atcccccacc agatccacct gggagagctg cacgccattc 3360
tgcggcggca ggaagatttt tacccattcc tgaaggacaa ccgggaaaag atcgagaaga 3420
tcctgacctt ccgcatcccc tactacgtgg gccctctggc caggggaaac agcagattcg 3480
cctggatgac cagaaagagc gaggaaacca tcaccccctg gaacttcgag gaagtggtgg 3540
acaagggcgc ttccgcccag agcttcatcg agcggatgac caacttcgat aagaacctgc 3600
ccaacgagaa ggtgctgccc aagcacagcc tgctgtacga gtacttcacc gtgtataacg 3660
agctgaccaa agtgaaatac gtgaccgagg gaatgagaaa gcccgccttc ctgagcggcg 3720
agcagaaaaa ggccatcgtg gacctgctgt tcaagaccaa ccggaaagtg accgtgaagc 3780
agctgaaaga ggactacttc aagaaaatcg agtgcttcga ctccgtggaa atctccggcg 3840
tggaagatcg gttcaacgcc tccctgggca cataccacga tctgctgaaa attatcaagg 3900
acaaggactt cctggacaat gaggaaaacg aggacattct ggaagatatc gtgctgaccc 3960
tgacactgtt tgaggacaga gagatgatcg aggaacggct gaaaacctat gcccacctgt 4020
tcgacgacaa agtgatgaag cagctgaagc ggcggagata caccggctgg ggcaggctga 4080
gccggaagct gatcaacggc atccgggaca agcagtccgg caagacaatc ctggatttcc 4140
tgaagtccga cggcttcgcc aacagaaact tcatgcagct gatccacgac gacagcctga 4200
cctttaaaga ggacatccag aaagcccagg tgtccggcca gggcgatagc ctgcacgagc 4260
acattgccaa tctggccggc agccccgcca ttaagaaggg catcctgcag acagtgaagg 4320
tggtggacga gctcgtgaaa gtgatgggcc ggcacaagcc cgagaacatc gtgatcgaaa 4380
tggccagaga gaaccagacc acccagaagg gacagaagaa cagccgcgag agaatgaagc 4440
ggatcgaaga gggcatcaaa gagctgggca gccagatcct gaaagaacac cccgtggaaa 4500
acacccagct gcagaacgag aagctgtacc tgtactacct gcagaatggg cgggatatgt 4560
acgtggacca ggaactggac atcaaccggc tgtccgacta cgatgtggac catatcgtgc 4620
ctcagagctt tctgaaggac gactccatcg acaacaaggt gctgaccaga agcgacaaga 4680
accggggcaa gagcgacaac gtgccctccg aagaggtcgt gaagaagatg aagaactact 4740
ggcggcagct gctgaacgcc aagctgatta cccagagaaa gttcgacaat ctgaccaagg 4800
ccgagagagg cggcctgagc gaactggata aggccggctt catcaagaga cagctggtgg 4860
aaacccggca gatcacaaag cacgtggcac agatcctgga ctcccggatg aacactaagt 4920
acgacgagaa tgacaagctg atccgggaag tgaaagtgat caccctgaag tccaagctgg 4980
tgtccgattt ccggaaggat ttccagtttt acaaagtgcg cgagatcaac aactaccacc 5040
acgcccacga cgcctacctg aacgccgtcg tgggaaccgc cctgatcaaa aagtacccta 5100
agctggaaag cgagttcgtg tacggcgact acaaggtgta cgacgtgcgg aagatgatcg 5160
ccaagagcga gcaggaaatc ggcaaggcta ccgccaagta cttcttctac agcaacatca 5220
tgaacttttt caagaccgag attaccctgg ccaacggcga gatccggaag cggcctctga 5280
tcgagacaaa cggcgaaacc ggggagatcg tgtgggataa gggccgggat tttgccaccg 5340
tgcggaaagt gctgagcatg ccccaagtga atatcgtgaa aaagaccgag gtgcagacag 5400
gcggcttcag caaagagtct atcctgccca agaggaacag cgataagctg atcgccagaa 5460
agaaggactg ggaccctaag aagtacggcg gcttcgacag ccccaccgtg gcctattctg 5520
tgctggtggt ggccaaagtg gaaaagggca agtccaagaa actgaagagt gtgaaagagc 5580
tgctggggat caccatcatg gaaagaagca gcttcgagaa gaatcccatc gactttctgg 5640
aagccaaggg ctacaaagaa gtgaaaaagg acctgatcat caagctgcct aagtactccc 5700
tgttcgagct ggaaaacggc cggaagagaa tgctggcctc tgccggcgaa ctgcagaagg 5760
gaaacgaact ggccctgccc tccaaatatg tgaacttcct gtacctggcc agccactatg 5820
agaagctgaa gggctccccc gaggataatg agcagaaaca gctgtttgtg gaacagcaca 5880
agcactacct ggacgagatc atcgagcaga tcagcgagtt ctccaagaga gtgatcctgg 5940
ccgacgctaa tctggacaaa gtgctgtccg cctacaacaa gcaccgggat aagcccatca 6000
gagagcaggc cgagaatatc atccacctgt ttaccctgac caatctggga gcccctgccg 6060
ccttcaagta ctttgacacc accatcgacc ggaagaggta caccagcacc aaagaggtgc 6120
tggacgccac cctgatccac cagagcatca ccggcctgta cgagacacgg atcgacctgt 6180
ctcagctggg aggcgacaaa aggccggcgg ccacgaaaaa ggccggccag gcaaaaaaga 6240
aaaagtaaga attcctagag ctcgctgatc agcctcgact gtgccttcta gttgccagcc 6300
atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 6360
cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 6420
ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagagaata gcaggcatgc 6480
tggggagcgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg 6540
ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc 6600
ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct 6660
tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt 6720
agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 6780
agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 6840
tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 6900
cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga 6960
tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 7020
caaactggaa caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg 7080
ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 7140
aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc 7200
gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 7260
ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 7320
aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt 7380
ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga 7440
aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 7500
atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 7560
caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 7620
cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 7680
tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 7740
tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac 7800
gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 7860
tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 7920
gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 7980
aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 8040
gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 8100
atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 8160
caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 8220
ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtggaag ccgcggtatc 8280
attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg 8340
agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 8400
aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 8460
catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctc 8508

Claims (10)

1.特异性靶向ApoE基因第二外显子的SgRNA，其特征在于，所述SgRNA的核苷酸序列如SEQ ID No:1所示。
2.含有权利要求1所述SgRNA的ApoE-CRISPR/Cas9载体。
3.根据权利要求2所述的ApoE-CRISPR/Cas9载体，其特征在于，所述ApoE-CRISPR/Cas9载体的核苷酸序列如SEQ ID No:2所示。
4.根据权利要求3所述的ApoE-CRISPR/Cas9载体，其特征在于，所述ApoE-CRISPR/Cas9载体包括U6基因启动子、SgRNA、CBh基因启动子和Cas9编码基因。
5.根据权利要求3所述的ApoE-CRISPR/Cas9载体，其特征在于，所述ApoE-CRISPR/Cas9载体按如下方法制备得到：
(1)用限制性内切酶消化pX330质粒，酶切后的质粒使用琼脂糖凝胶分离，用胶回收试剂盒纯化回收酶切产物；
(2)将权利要求1所述的SgRNA序列按如下程序退火：
37℃30min
95℃5min然后以5℃/min的速率降至25℃；
(3)将步骤(1)得到的酶切产物和步骤(2)退火后的SgRNA序列使用连接酶进行连接；
(4)用PlasmidSafe核酸外切酶处理步骤(3)得到的体系，去除错误连接的质粒；
(5)将质粒转化到感受态细胞中进行培养；
(6)从步骤(5)培养的感受态细胞中提取质粒进行测序，确定载体构建成功。
6.权利要求2-5任一所述的ApoE-CRISPR/Cas9载体在构建ApoE基因敲除的哺乳动物模型中的应用。
7.根据权利要求6所述的应用，其特征在于，包括如下步骤：
(1)将ApoE-CRISPR/Cas9载体转化至哺乳动物的胎儿成纤维细胞中；
(2)使用G418抗生素对步骤(1)得到的成纤维细胞进行抗性筛选，将具有抗性的成纤维细胞进行基因测序，保留成功转基因的成纤维细胞；
(3)将步骤(2)得到的成纤维细胞的细胞核移植到去核的哺乳动物卵母细胞中培养至囊胚阶段；
(4)将步骤(3)得到的囊胚移植到代孕哺乳动物中进行饲养，生产；
(5)提取步骤(4)生产的哺乳动物的基因组，利用PCR引物进行扩增，进行基因型鉴定。
8.根据权利要求7所述的应用，其特征在于，步骤(1)～(5)中所述哺乳动物为巴马小型猪。
9.根据权利要求7所述的应用，其特征在于，步骤(5)中所述PCR引物为：正向引物为5’-gcagggcgtgagcattagat-3’，反向引物为5’-gagttggcgacaaggacagaa-3’。
10.权利要求2-5任一所述的ApoE-CRISPR/Cas9载体在制备ApoE基因敲除试剂盒中的应用。
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