CN111778278A - 一种Slfn4缺失的动脉粥样硬化模型小鼠的构建方法及其应用 - Google Patents
一种Slfn4缺失的动脉粥样硬化模型小鼠的构建方法及其应用 Download PDFInfo
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Abstract
本发明涉及一种Slfn4缺失的动脉粥样硬化模型小鼠的构建方法及其应用。本发明将Slfn4基因敲除通过CRISPR‑Cas9系统引入ApoE基因缺失遗传背景的动脉粥样硬化小鼠,回交和杂交后进行基因型筛选,获得了双基因缺失的纯合子模型小鼠ApoE–/–Slfn4–/–。Slfn4缺失动脉粥样硬化模型小鼠(ApoE–/–Slfn4–/–)较ApoE–/–小鼠动脉粥样硬化斑块显著减少,症状显著减轻,表明Slfn4基因具有促进主动脉粥样硬化斑块形成的作用。针对Slfn4上述功能,Slfn4可以作为药物靶标筛选治疗动脉粥样硬化疾病的药物;Slfn4抑制剂能够用于制备治疗动脉粥样硬化疾病的药物。
Description
技术领域
本发明涉及一种Slfn4缺失的动脉粥样硬化模型小鼠的构建方法及其应用,属于基因工程和遗传修饰技术领域。
背景技术
动脉粥样硬化为血管阻塞性疾病的主要原因,由其所致脑梗死、心肌梗死等是我国首要致残、致死性疾病。动脉粥样硬化的形成、发展与单核巨噬细胞密切相关,后者吞噬脂质形成泡沫细胞沉积于血管壁、释放细胞因子并招募免疫细胞形成慢性炎症,是动脉粥样硬化的病理基础。
慢性炎症是导致动脉粥样硬化的核心因素。免疫细胞不断迁移到血管壁,与脂质等共同聚集导致动脉粥样硬化斑块的形成。斑块破裂后血栓形成、从而导致脑梗死、冠状动脉粥样硬化性心脏病等致残、致死性后果。而动脉粥样硬化斑块中的免疫细胞主要是巨噬细胞,后者来源于循环血液中单核细胞,吞噬脂质后形成泡沫细胞。单核巨噬细胞对动脉粥样硬化形成的作用体现在两个方面:第一、动脉粥样硬化形成早期,血液中的单核细胞通过内皮间隙,在内膜下分化为巨噬细胞;巨噬细胞通过清道夫受体CD36结合并吞入氧化型低密度脂蛋白(Ox-LDL),酪氨酸激酶Lyn发生磷酸化,磷酸化的Lyn级联激活JNK1/2或Vav等,促进Ox-LDL摄取,导致细胞内脂质堆积,形成泡沫细胞。第二、进入内膜后,单核细胞在巨噬细胞集落刺激因子(M-CSF)等因子作用下分化成两类巨噬细胞:经典促炎型(M1)和抑炎型(M2)巨噬细胞,分别集中于不稳定和稳定斑块中,并对动脉粥样硬化的病理进程发挥调节作用。二者在斑块中呈动态变化,巨噬细胞数量和分型影响斑块的进展。因此,减轻巨噬细胞脂质蓄积和炎症反应的调控措施对防治动脉粥样硬化性心血管疾病具有重要意义。
髓样分化因子Slfn4特异且高丰度表达于单核巨噬细胞,而单核巨噬细胞对动脉粥样硬化的有着关键影响,但Slfn4在高脂血症条件下对单核巨噬细胞的分化、泡沫细胞形成的作用还缺乏研究。
发明内容
本发明的目的在于提供一种Slfn4缺失的动脉粥样硬化模型小鼠的构建方法,并通过该模型探索Slfn4基因在动脉粥样硬化疾病发生、发展中的作用,提供Slfn4作为靶标用于筛选治疗动脉粥样硬化疾病的药物以及Slfn4抑制剂在制备治疗动脉粥样硬化疾病药物中的应用。
为了实现上述目的,本发明采用以下技术方案:
本发明提供一种Slfn4缺失的动脉粥样硬化模型小鼠的构建方法:先将ApoE–/–小鼠的Slfn4基因敲除,获得F0代小鼠;再对F0代小鼠进行基因型鉴定选择Slfn4基因敲除的F0代ApoE–/–背景小鼠,并将其与ApoE–/–小鼠进行回交获得F1代Slfn4基因杂合子小鼠;最后将F1代Slfn4基因杂合子小鼠进行自交获得F2代小鼠,对F2代小鼠进行基因型鉴定筛选,获得Slfn4双等位基因敲除且ApoE双等位基因敲除的纯合子个体即为Slfn4基因敲除动脉粥样硬化模型小鼠。
所述F0代小鼠的获得具体包括如下步骤:
1)靶向小鼠Slfn4基因的sgRNA的选择:CRISPR-Cas9系统中sgRNA作用位点位于Slfn4基因的2号外显子区,sgRNA作用位点DNA靶序列分别如下所示:
Slfn4-sgRNA1:5’-AGGTGCAGGATACCAGGCAA GGG-3’;
Slfn4-sgRNA2:5’-AAGCCGAATCAGAGAGGTCC GGG-3’;
Slfn4-sgRNA3:5’-CTCAGTTGAACTGAAAGCAG CGG-3’;
2)引物设计:根据步骤1)获得的sgRNA靶序列设计3条引物,引物序列如下所示:
Slfn4-IVT-1:5’-TTCTAATACGACTCACTATAGGAGGTGCAGGATACCA
GGCAAGTTTTAGAGCTAGA-3’;
Slfn4-IVT-2:5’-TTCTAATACGACTCACTATAGGAAGCCGAATCAGAGA
GGTCCGTTTTAGAGCTAGA-3’;
Slfn4-IVT-3:5’-TTCTAATACGACTCACTATAGGCTCAGTTGAACTGAA
AGCAGGTTTTAGAGCTAGA-3’;
Slfn4-IVT-4:5’-AAAAAAGCAC CGACTCGGTG CC-3’;
3)合成双链DNA片段:将步骤2)中的Slfn4-IVT-1、Slfn4-IVT-2、Slfn4-IVT-3分别与Slfn4-IVT-4配对,以pX330质粒为模板,进行聚合酶链式反应并对获得的DNA扩增产物进行纯化。
4)获得sgRNA和Cas9 mRNA:分别以步骤3)中的双链DNA片段为模版,体外转录并纯化,获得sgRNA1、sgRNA2和sgRNA 3;以AgeI单酶切线性化的pST1374-Cas9质粒为模版,体外转录并纯化,获得Cas9 mRNA;
5)获得Slfn4基因敲除的F0代小鼠:将步骤4)获得的3个sgRNA和Cas9 mRNA等比例注射由ApoE–/–小鼠精子和C57BL/6小鼠卵细胞体外受精形成的受精卵细胞,获得Slfn4基因敲除的受精卵细胞并其移植至假孕雌鼠,出生小鼠即为Slfn4基因敲除的F0代小鼠。
所述步骤1)中,通过MGI数据库(http://www.informatics.jax.org/)获取Slfn4基因序列,通过在线软件CRISPOR在Slfn4基因Exon2区段筛选具有较高特异性的3个特异性位点作为sgRNA的靶序列。
所述步骤4)中,Cas9 mRNA、sgRNA1、sgRNA2和sgRNA3的注射浓度均为50-100ng/μL。
本发明将Slfn4基因敲除通过CRISPR-Cas9系统引入ApoE基因缺失遗传背景的动脉粥样硬化小鼠,回交和杂交后进行基因型筛选,获得了双基因缺失的纯合子模型小鼠ApoE–/–Slfn4–/–。采用本发明构建方法获得的ApoE–/–Slfn4–/–模型小鼠可正常繁育和哺乳,且免疫系统和野生型小鼠无显著性差异,能够为后续Slfn4在动脉粥样硬化疾病发生、发展中的作用及动脉粥样硬化诊断治疗等方面的研究提供充足、可靠的ApoE–/–Slfn4–/–模型小鼠双基因缺失的纯合模型小鼠。
通过本发明提供的构建方法得到的Slfn4缺失的动脉粥样硬化模型小鼠为研究Slfn4基因在动脉粥样硬化疾病发生、发展中的作用奠定了基础。具体的,Slfn4缺失的动脉粥样硬化模型小鼠能够用于研究Slfn4在动脉粥样硬化的形成与修复中的作用,或者用于研究动脉粥样硬化的诊断治疗方法。
为了阐明Slfn4基因与动脉粥样硬化疾病的关系以及Slfn4如何影响动脉粥样硬化病程的发生和发展,本发明对获得的Slfn4缺失的动脉粥样硬化模型小鼠进行进一步的实验研究:
对Slfn4缺失动脉粥样硬化模型小鼠(基因型为ApoE–/–Slfn4–/–)和对照小鼠(基因型为ApoE–/–)进行高脂饲料喂养12周后,将其心脏组织使用4%多聚甲醛固定过夜,并对主动脉根部及主动脉区域进行油红O染色分析,对两组小鼠主动脉根部或主动脉区域斑块面积进行统计。结果表明,与对照小鼠相比,在ApoE–/–Slfn4–/–模型小鼠中,动脉粥样硬化斑块显著减少,动脉粥样硬化症状显著减轻,表明Slfn4缺失动脉粥样硬化模型小鼠构建是成功的,且Slfn4缺失减缓了动脉粥样硬化的病变程度。
本发明确定了Slfn4具有促进主动脉粥样硬化斑块形成的作用,主要体现在Slfn4能够促进主动脉粥样硬化的发生、发展。
针对Slfn4上述作用,本发明提供Slfn4作为药物靶标在筛选治疗动脉粥样硬化疾病的药物中的应用。
针对Slfn4上述作用,本发明提供Slfn4抑制剂在制备治疗动脉粥样硬化疾病药物中的应用。所述的Slfn4抑制剂优选Slfn4基因的siRNA、Slfn4抗体或其他能够抑制Slfn4表达的抑制剂。
本发明在动脉粥样硬化小鼠中实现了Slfn4基因的敲除,并获得了Slfn4缺失且能正常存活的动脉粥样硬化模型小鼠。具有非常重要的基础研究和实际应用价值,也为开发动脉粥样硬化治疗的新方案提供了新的思路。本发明确定了3个针对小鼠Slfn4基因的特异性打靶位点,实验数据表明该特异性位点具有较高的剪切效率;获得了大片段缺失的Slfn4基因敲除动脉粥样硬化小鼠,一方面保证了Slfn4基因能够彻底失去功能,另一方面方便后续子代小鼠大批量的Slfn4基因型检测。本发明阐明了Slfn4基因在动脉粥样硬化疾病发生、发展中的作用:Slfn4基因具有促进主动脉粥样硬化斑块形成的作用。针对Slfn4上述功能,Slfn4可以作为药物靶标筛选治疗动脉粥样硬化疾病的药物;Slfn4抑制剂能够用于制备治疗动脉粥样硬化疾病的药物。这为深入开展动脉粥样硬化的研究开启了暂新的方向,也为动脉粥样硬化的临床治疗提供了更多的选择,具有非常重要的理论和实际意义。
附图说明
图1为ApoE–/–遗传背景小鼠中针对Slfn4基因敲除打靶sgRNA设计方案图;
图2为ApoE–/–遗传背景Slfn4基因敲除F1代小鼠荧光PCR基因型检测图;
图3为ApoE–/–遗传背景Slfn4基因敲除F2代小鼠Sanger测序图;
图4为ApoE–/–Slfn4–/–模型小鼠和对照APOE–/–小鼠饲喂高脂饲料12周期间体重分析图;
图5为ApoE–/–Slfn4–/–模型小鼠和对照APOE–/–小鼠饲喂高脂饲料12周后血清生化指标检测图;
图6为ApoE–/–Slfn4–/–模型小鼠和对照APOE–/–小鼠饲喂高脂饲料12周后主动脉根部冰冻切片油红O和HE染色分析图;
图7为ApoE–/–Slfn4–/–模型小鼠和对照APOE–/–小鼠饲喂高脂饲料12周后主动脉油红O染色分析图。
具体实施方式
下面结合具体实施例对本发明做进一步详细说明。ApoE基因敲除小鼠(基因型为APOE–/–)由美国Jackson Laboratory实验室购买,该小鼠高脂饲料喂养数周后患动脉粥样硬化,是研究动脉粥样硬化常用的经典工具小鼠。所使用的高脂饲料配方参考ResearchDiets公司编号D12108C。使用100mL 100%异丙醇溶解0.5g油红O制成油红O储夜并置于4℃冰箱保存,使用前将储夜和蒸馏水按照3:2进行稀释配制成油红O工作液并进行过滤,油红O工作液正常情况下呈现酒红色且无沉淀。pX330质粒DNA购于Addgene;pT7-3×Flag-hCas9质粒由黄军就教授馈赠。其他实验用品未经说明,均为市售商品。
实施例1
本实施例中的Slfn4缺失的动脉粥样硬化模型小鼠的构建方法通过以下步骤实现:
一、靶向小鼠Slfn4基因的sgRNA的选择和设计
通过小鼠基因组数据库MGI(http://www.informatics.jax.org/)获取Slfn4基因序列,根据CRISPR/Cas9系统原理,使用CRISPOR在线设计软件(http://crispor.tefor.net/crispor.cgi)在小鼠Slfn4基因的靶位点Exon2(exon ID:ENSMUSE00000108009)区域挑选三个特异性位点作为sgRNA的靶序列(如图1所示),如下所示:
Slfn4-sgRNA1:5’-AGGTGCAGGATACCAGGCAA GGG-3’(SEQ ID NO.1);
Slfn4-sgRNA2:5’-AAGCCGAATCAGAGAGGTCC GGG-3’(SEQ ID NO.2);
Slfn4-sgRNA3:5’-CTCAGTTGAACTGAAAGCAG CGG-3’(SEQ ID NO.3)。
二、sgRNA体外转录
1.根据步骤一获得的sgRNA靶序列设计4条引物,引物序列如下所示:
Slfn4-IVT-1:5’-TTCTAATACGACTCACTATAGGAGGTGCAGGATACCA
GGCAAGTTTTAGAGCTAGA-3’(SEQ ID NO.4);
Slfn4-IVT-2:5’-TTCTAATACGACTCACTATAGGAAGCCGAATCAGAGA
GGTCCGTTTTAGAGCTAGA-3’(SEQ ID NO.5);
Slfn4-IVT-3:5’-TTCTAATACGACTCACTATAGGCTCAGTTGAACTGAA
AGCAGGTTTTAGAGCTAGA-3’(SEQ ID NO.6);
Slfn4-IVT-4:5’-AAAAAAGCAC CGACTCGGTG CC-3’(SEQ ID NO.7);
交由上海百力格生物技术有限公司进行上述引物合成。
2.使用5’端包含T7启动子序列的上述引物进行聚合酶链式反应,获得具有T7启动子序列的双链DNA片段。
聚合酶链式反应体系:模板(pX330载体,购于Addgene)加10ng;10μM上游引物Slfn4-IVT-1、Slfn4-IVT-2和Slfn4-IVT-3分别加2μL;10μM下游通用引物(Slfn4-IVT-4)加2μL;2×Taq Master Mix(购于Vazyme,P111-01)加25μL;补充H2O至总体积50μL。
聚合酶链式反应程序:94℃预变性5Min;94℃变性30s,60℃退火30s,72℃延伸30s;72℃终延伸10Min;循环数为30次。之后使用Invitrogen PCR回收试剂盒(Invitrogen,K220001)对聚合酶链式反应产物进行分离和纯化。
3.体外转录获得sgRNA:使用NEB试剂盒(NEB,E2050S)进行体外转录,具体步骤如下:按照试剂盒操作说明,配制反应体系;依次加入NTP Buffer Mix 10μL,模板DNA1μg,T7RNA Polymerase Mix 2μL,补充H2O至总体积30μL;置于37℃反应4h,获得体外转录产物。之后使用Ambion RNA纯化试剂盒(Ambion,AM1909)回收条带即可获得sgRNA1、sgRNA2和sgRNA3。
三、Cas9 DNA体外转录
1.获得Cas9体外转录模板DNA:使用高纯度质粒抽提试剂盒(天根生化科技(北京)有限公司,DP116)提取Cas9载体质粒DNA,然后采用以下体系使用AgeI限制性内切酶(NEB,R3552S)酶切质粒DNA:质粒DNA加入10μg;10×CutSmart Buffer加入20μL;AgeI限制性内切酶加入2μL;补充H2O至总体积200μL。将上述反应体系置于37℃恒温箱反应12h。通过琼脂糖凝胶电泳检测酶切效果,酶切产物电泳后呈现单一条带则表明载体质粒DNA线性化完成。使用DNA回收试剂盒(天根生化科技(北京)有限公司,DP205)对酶切产物进行分离和纯化,即获得Cas9体外转录模板DNA。
2.体外转录获得Cas9 mRNA:使用Invitrogen试剂盒(invitrogen,AMB1345-5)按照如下步骤配制反应体系:依次加入T7 2*NTP/ARCA 10μL,10*T7 Reaction Buffer 2μL,模板DNA 1μg,T7 Enzyme Mix 2μL,补充H2O(Invitrogen,10977015)至总体积20μL;置于37℃反应孵育2h,然后加入1μL TURBO DNase,充分混匀后置于37℃反应孵育15Min;之后向上述反应产物中按照以下顺序配制加尾反应体系,mMESSAGE mMACHINE T7 Ultra reaction产物20μL,H2O(Invitrogen,10977015)36μL,5×E-PAP Buffer 20μL,25mM MnCl2 10μL,ATPSolution 10μL,E-PAP 4μL,充分混匀后,置于37℃反应孵育45Min。获得体外转录产物以后,使用Ambion RNA纯化试剂盒(Ambion,AM1909)回收Cas9 mRNA。
四、受精卵显微注射
1.制备单细胞受精卵:通过对8周龄C57BL/6雌鼠注射孕马血清和人绒毛膜促性腺激素进行超排卵,之后将2只C57BL/6雌鼠和一只ApoE–/–背景雄鼠合笼。第四天上午检栓。CO2处死见栓0.5天的C57BL/6雌鼠,剪出输卵管,用显微镊取出成团的卵子,透明质酸酶消化后,挑选形态饱满、胞质均匀的胚胎于M16中培养。
2.显微注射受精卵:将3条sgRNA和Cas9 mRNA使用无RNA酶的超纯水(Invitrogen,10977015)稀释至终浓度均为50ng/μL,混合后通过显微注射系统注射入ApoE–/–背景小鼠受精卵细胞,置于37℃、5%二氧化碳培养箱恢复1h,挑选存活的细胞,将其移植入假孕ICR小鼠雌鼠输卵管中。20天以后即可获得F0代小鼠。
五、F0代小鼠的基因型鉴定
通过荧光聚合酶链式反应对F0代小鼠进行基因型检测。剪取出生一周的F0代小鼠鼠尾,抽提其基因组DNA作为模板DNA,使用检测引物进行荧光聚合酶链式反应,检测引物序列如下所示:
Slfn4-GeneTy-F:5’-TCGGAGAGAAGACTAGGGATTCA-3’(SEQ ID NO.8,5‘端使用FAM荧光进行标记);
Slfn4-GeneTy-R:5’-GCAGAAACAGTTTGAGGGAGG-3’(SEQ ID NO.9);
PCR反应体系为:鼠尾DNA模板20ng,上游和下游引物各加2μL,2×Taq Master Mix(Vazyme,P111-01)加25μL,补充H2O至总体积50μL。PCR反应程序为:94℃预变性5Min;94℃变性30s,60℃退火30s,72℃延伸30s,循环数为30次;72℃终延伸10Min。以Slfn4野生型鼠尾DNA为模板的PCR产物为574bp。取2μL PCR产物首先进行琼脂糖电泳检测,如果电泳呈现明显的条带,则将PCR产物稀释200倍并与甲酰胺、标准分子量内参DNA混合,变性后置于测序仪上进行毛细管电泳,结果使用Genemapper软件进行数据分析,直接获得PCR产物的大小,与野生型对照的PCR产物大小进行比较后,得出碱基插入或缺失的具体数目。与野生型扩增产物对照进行比较,发现2只F0代小鼠(分别命名为#1和#2)的扩增片段与野生型相比,均发生了缺失。
结果如图2所示,获得的两只小鼠均为杂合子,其中#1小鼠Slfn4基因位点一条染色体缺失189bp,另外一条染色体为野生型;#2小鼠Slfn4基因位点一条染色体缺失174bp,另外一条染色体为野生型。
通过TA克隆检测小鼠基因型。使用引物序列为:
Slfn4-GeneTy-F:5’-TCGGAGAGAAGACTAGGGATTCA-3’(SEQ ID NO.8);
Slfn4-GeneTy-R:5’-GCAGAAACAGTTTGAGGGAGG-3’(SEQ ID NO.9);
其中Slfn4野生型DNA模板扩增片段长度为574bp(如SEQ ID NO.10所示),PCR反应体系与程序同上,分别取2μL#1小鼠和#2小鼠的PCR扩增产物用于TA克隆酶连反应(天根生化科技(北京)有限公司,DP205),将酶连产物转化DH5α感受态细胞并置于37℃培养箱培养过夜,之后挑取单克隆进行培养并送样进行基因型测序鉴定。2只F0小鼠均进行测序鉴定,测序结果如图3所示,A和B分别为#1和#2Slfn4基因敲除小鼠的基因型;与图2荧光聚合酶链式反应结果一致。
将2只F0代小鼠进行回交和自交获得Slfn4基因敲除ApoE–/–小鼠纯合子。具体为:将F0小鼠与ApoE单基因敲除小鼠回交获得F1代小鼠,在F1代小鼠中挑选杂合子自交获得F2代小鼠,F2代经检测获得Slfn4基因敲除纯合子,此小鼠即为ApoE–/–遗传背景的Slfn4缺失模型小鼠。同时保留筛选得到的杂合子小鼠,作为后续繁殖的亲本。
实施例2
将实施例1中获得的Slfn4缺失的动脉粥样硬化模型小鼠(基因型为ApoE–/–Slfn4–/–)和对照小鼠(基因型为ApoE–/–)进行高脂饲喂12周。期间每周定时监测体重,12周后使用全自动生化分析仪(ADVIA2400,SIEMENS)对血清总胆固醇(TC)、甘油三脂(TG)、高密度脂蛋白(HDL)和低密度脂蛋白(LDL)等生化指标进行检测,并对数据进行统计分析。
实验结果表明Slfn4缺失动脉粥样硬化模型小鼠与对照小鼠相比体重变化无显著差异(如图4所示),血清生化指标无显著差异(如图5所示)。
实施例3
对Slfn4缺失的动脉粥样硬化模型小鼠(基因型为ApoE–/–Slfn4–/–)和对照小鼠(基因型为ApoE–/–)进行高脂饲料喂养12周后,将其心脏组织使用4%多聚甲醛固定过夜并使用OCT进行包埋,将主动脉根部制成10μm冰冻切片,然后进行油红O染色和HE染色,并对两组小鼠主动脉根部斑块面积进行统计。
结果如图6所示,Slfn4缺失的动脉粥样硬化模型小鼠较对照小鼠主动脉根部斑块面积明显减少(如图6A所示)。两组小鼠主动脉根部斑块面积统计结果(如图6B所示)表明Slfn4缺失动脉粥样硬化模型小鼠(ApoE–/–Slfn4–/–)与动脉粥样硬化模型小鼠(ApoE–/–)相比斑块面积显著减少,说明Slfn4缺失减轻了动脉粥样硬化的病变程度。
本发明的构建方法获得的Slfn4缺失的动脉粥样硬化模型小鼠可用于研究Slfn4对动脉粥样硬化的形成与修复的作用。本发明的构建方法获得的Slfn4缺失的动脉粥样硬化模型小鼠也可以用于研究动脉粥样硬化诊断治疗方法。
实施例4
对Slfn4缺失的动脉粥样硬化模型小鼠(基因型为ApoE–/–Slfn4–/–)和对照小鼠(基因型为ApoE–/–)进行高脂饲料喂养12周后,将其心脏组织使用4%多聚甲醛固定过夜然后进行油红O染色,并对两组小鼠主动脉区域斑块面积进行统计。
结果如图7所示,Slfn4缺失的动脉粥样硬化模型小鼠(基因型为ApoE–/–Slfn4–/–)较对照小鼠(基因型为ApoE–/–)主动脉区域的斑块明显减少(如图7A所示)。对两组小鼠主动脉区域斑块面积进行统计分析(如图7-B所示)显示Slfn4缺失动脉粥样硬化模型小鼠(ApoE–/–Slfn4–/–)较对照小鼠(基因型为ApoE–/–)的主动脉区域斑块面积显著减少。
综合分析上述实验结果,与对照小鼠(基因型为ApoE–/–)相比,Slfn4缺失的动脉粥样硬化模型小鼠(基因型为ApoE–/–Slfn4–/–)的动脉粥样斑块显著减少,动脉粥样硬化症状明显减轻。表明本申请中Slfn4缺失动脉粥样硬化模型小鼠构建是成功的,而且Slfn4缺失会减轻动脉粥样硬化的病变程度,这表明Slfn4基因具有促进主动脉粥样硬化斑块形成的作用。针对Slfn4上述功能,Slfn4可以作为药物靶标筛选治疗动脉粥样硬化疾病的药物;Slfn4抑制剂能够用于制备治疗动脉粥样硬化疾病的药物。为研制防治脉粥样硬化药物提供了新靶点和新策略。
SEQUENCE LISTING
<110> 新乡医学院
<120> 一种Slfn4缺失的动脉粥样硬化模型小鼠的构建方法及其应用
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213> 人工序列
<221> Slfn4-sgRNA1
<400> 1
aggtgcagga taccaggcaa ggg 23
<210> 2
<211> 23
<212> DNA
<213> 人工序列
<221> Slfn4-sgRNA2
<400> 2
aagccgaatc agagaggtcc ggg 23
<210> 3
<211> 23
<212> DNA
<213> 人工序列
<221> Slfn4-sgRNA3
<400> 3
ctcagttgaa ctgaaagcag cgg 23
<210> 4
<211> 56
<212> DNA
<213> 人工序列
<221> Slfn4-IVT-1
<400> 4
ttctaatacg actcactata ggaggtgcag gataccaggc aagttttaga gctaga 56
<210> 5
<211> 56
<212> DNA
<213> 人工序列
<221> Slfn4-IVT-2
<400> 5
ttctaatacg actcactata ggaagccgaa tcagagaggt ccgttttaga gctaga 56
<210> 6
<211> 56
<212> DNA
<213> 人工序列
<221> Slfn4-IVT-3
<400> 6
ttctaatacg actcactata ggctcagttg aactgaaagc aggttttaga gctaga 56
<210> 7
<211> 22
<212> DNA
<213> 人工序列
<221> Slfn4-IVT-4
<400> 7
aaaaaagcac cgactcggtg cc 22
<210> 8
<211> 23
<212> DNA
<213> 人工序列
<221> Slfn4-GeneTy-F
<400> 8
tcggagagaa gactagggat tca 23
<210> 9
<211> 21
<212> DNA
<213> 人工序列
<221> Slfn4-GeneTy-R
<400> 9
gcagaaacag tttgagggag g 21
<210> 10
<211> 574
<212> DNA
<213> 小鼠
<221> Slfn4基因片段
<400> 10
tcggagagaa gactagggat tcaatgaaag atagccaact gagaagaaaa gaagccaaaa 60
gtatattaca ggctgtgtgc accctgctga attctggagg gggtgtggtc aaggctcaca 120
ttaaaaatca aaactacagc ttcaccagag atggaatggg actggatttg gtaaatccct 180
tgcctggtat cctgcacctt cctcatgact atctagactt catgcagtac aacgactact 240
ttttcgtttt tgtgaaacca tggaagccga atcagagagg tccggggatc gccacctgga 300
aaaccaactt gtacaagaga atcttctcat tctcagttga actgaaagca gcggatgcag 360
tgcagttcct caaatccaga ccaagttccc atggaaaaac agtctgtaat gaaacactaa 420
atgaatgtct ttccttattt aacagagact ggcttgccta tgaggagaca ttctgtttca 480
ctaaatccac acatgctgaa gtaaaattga ctcctaagga aaagatttct cctaaggaaa 540
agattttaga gctcctccct caaactgttt ctgc 574
Claims (9)
1.一种Slfn4缺失的动脉粥样硬化模型小鼠的构建方法,其特征在于,先将ApoE–/–小鼠的Slfn4基因敲除,获得F0代小鼠;再对F0代小鼠进行基因型鉴定选择Slfn4基因敲除的F0代ApoE–/–背景小鼠,并将其与ApoE–/–小鼠进行回交获得F1代Slfn4基因杂合子小鼠;最后将F1代Slfn4基因杂合子小鼠进行自交获得F2代小鼠,对F2代小鼠进行基因型鉴定筛选,获得Slfn4双等位基因敲除且ApoE双等位基因敲除的纯合子个体即为Slfn4基因敲除动脉粥样硬化模型小鼠。
2.根据权利要求1所述的Slfn4缺失的动脉粥样硬化模型小鼠的构建方法,其特征在于,所述F0代小鼠的获得是采用CRISPR/Cas9技术将ApoE–/–小鼠的Slfn4基因敲除。
3.根据权利要求2所述的Slfn4缺失的动脉粥样硬化模型小鼠的构建方法,其特征在于,所述F0代小鼠的获得包括以下步骤:
1)靶向小鼠Slfn4基因的sgRNA选择:CRISPR-Cas9系统中sgRNA作用位点位于Slfn4基因的2号外显子区,sgRNA作用位点DNA靶序列分别如下所示:
Slfn4-sgRNA1:5’-AGGTGCAGGATACCAGGCAA GGG-3’;
Slfn4-sgRNA2:5’-AAGCCGAATCAGAGAGGTCC GGG-3’;
Slfn4-sgRNA3:5’-CTCAGTTGAACTGAAAGCAG CGG-3’;
2)引物设计:根据步骤1)获得的sgRNA靶序列设计4条引物,引物序列如下所示:
Slfn4-IVT-1:5’-TTCTAATACGACTCACTATAGGAGGTGCAGGATACCAGGCAAGTTTTAGAGCTAGA-3’;
Slfn4-IVT-2:5’-TTCTAATACGACTCACTATAGGAAGCCGAATCAGAGAGGTCCGTTTTAGAGCTAGA-3’;
Slfn4-IVT-3:5’-TTCTAATACGACTCACTATAGGCTCAGTTGAACTGAAAGCAGGTTTTAGAGCTAGA-3’;
Slfn4-IVT-4:5’-AAAAAAGCAC CGACTCGGTG CC-3’;
3)合成双链DNA片段:将步骤2)中的Slfn4-IVT-1、Slfn4-IVT-2、Slfn4-IVT-3分别与Slfn4-IVT-4配对,以pX330质粒为模板,进行聚合酶链式反应并对获得的DNA扩增产物进行纯化;
4)获得sgRNA和Cas9 mRNA:分别以步骤3)中双链DNA片段为模版,体外转录并纯化,获得sgRNA1、sgRNA2和sgRNA3;以AgeI单酶切线性化的pST1374-Cas9质粒为模版,体外转录并纯化,获得Cas9 mRNA;
5)获得Slfn4缺失的F0代小鼠:将步骤4)获得的3个sgRNA和Cas9 mRNA等比例注射由ApoE–/–小鼠精子和C57BL/6小鼠卵细胞体外受精产生的受精卵细胞,获得Slfn4基因敲除的受精卵细胞并其移植至假孕雌鼠,出生的小鼠即为Slfn4基因敲除的F0代小鼠。
4.根据权利要求1所述的Slfn4缺失的动脉粥样硬化模型小鼠的构建方法,其特征在于,Slfn4基因敲除小鼠的基因型鉴定的引物如下所示:
Slfn4-GeneTy-F:5’-TCGGAGAGAAGACTAGGGATTCA-3’;
Slfn4-GeneTy-R:5’-GCAGAAACAGTTTGAGGGAGG-3’。
5.根据权利要求4所述的Slfn4缺失的动脉粥样硬化模型小鼠的构建方法,其特征在于,采用琼脂糖凝胶电泳对Slfn4基因型进行分析。
6.利用如权利要求1所述的构建方法得到的Slfn4缺失的动脉粥样硬化模型小鼠在研究动脉粥样硬化疾病发生、发展方面的应用。
7.利用如权利要求1所述的构建方法得到的Slfn4缺失的动脉粥样硬化模型小鼠在研究动脉粥样硬化诊断治疗方面的应用。
8.Slfn4作为药物靶标在筛选治疗动脉粥样硬化疾病药物中的应用。
9.Slfn4抑制剂在制备治疗动脉粥样硬化疾病药物中的应用。
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