CN107022572A - Ldl受体基因敲除的基因工程仓鼠 - Google Patents
Ldl受体基因敲除的基因工程仓鼠 Download PDFInfo
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- CN107022572A CN107022572A CN201610069985.2A CN201610069985A CN107022572A CN 107022572 A CN107022572 A CN 107022572A CN 201610069985 A CN201610069985 A CN 201610069985A CN 107022572 A CN107022572 A CN 107022572A
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
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Abstract
本发明提供了一种制备基因工程仓鼠的方法,包括将该仓鼠体内的LDL受体基因敲除。在优选实施方案中,所述LDL受体基因通过CRISPR/CAS9基因编辑技术敲除。该基因工程仓鼠表现出明显的血脂升高,可以直接用于高脂血症和动脉硬化研究,是和人类家族性高胆固醇血症高度相似的不可多得的小动物模型。本发明还提供了利用该基因工程仓鼠筛选药物的方法。
Description
技术领域
本发明涉及基因工程动物领域,更具体地说,涉及通过CRISPR/CAS9基因编辑技术构建的LDL受体基因敲除的仓鼠。
背景技术
高胆固醇血症(hypercholesterolemia)是动脉粥样硬化和心血管疾病最重要的独立危险因子之一,并与诸多遗传和环境因素有相互作用。家族型高胆固醇血症(familialhypercholesterolemia)是原发性高胆固醇血症之一,其发病机理主要是低密度脂蛋白(low density lipoprotein,LDL)受体的基因突变功能下降或丧失,血浆LDL清除减慢,胆固醇水平升高,导致早发性心脑血管疾病并常伴有皮肤和肌腱黄色瘤(1,2)。
选择恰当的疾病动物模型是研究人类疾病发生、发展及转归,药物筛选及药理学研究的必然手段。小鼠因其体积小、繁殖快、费用低,更因为有多种基因工程模型,因此广泛用于各种疾病的实验研究。小鼠对动脉粥样硬化不易感,以往基本不用于动脉粥样硬化的研究;直至载脂蛋白E(apolipoprotein E,apoE)(3)和低密度脂蛋白受体(low densitylipoprotein receptor,LDL-R)基因敲除小鼠(4)的问世,小鼠迅速变成了代谢性心血管病研究领域中最常用的模式动物。apoE和LDL-R纯合子KO小鼠可以自发动脉粥样硬化病变(5),高脂饲料可加速这种病变的发生。然而由于小鼠脂代谢与人类差异较大,对药物的敏感性不同,一些基于小鼠取得的研究结果在临床应用上存在一定争议(6)。此外,小鼠动脉粥样病变多发于主动脉和流出道,极少累及冠脉和脑动脉。因此,基于apoE KO和LDL-R KO小鼠高胆固醇血症的心脑血管并发症的研究,缺乏冠状动脉和脑动脉病变的基础病因和病生理基础,不能模拟人类冠心病和中风的自然发病过程,有明显局限性。
发明内容
本发明一方面提供了一种基因工程仓鼠,其中该仓鼠体内的LDL受体基因被敲除。
在一些实施方案中,该仓鼠为LDL受体基因敲除纯合子。在另一些实施方案中,该仓鼠为LDL受体基因敲除杂合子。
在一些实施方案中,该仓鼠的血脂水平显著升高。
本发明另一方面提供了一种制备基因工程仓鼠的方法,包括:将该仓鼠体内的LDL受体基因敲除。
在优选实施方案中,所述LDL受体基因通过CRISPR/CAS9基因编辑技术被敲除。在一些具体实施方案中,其中所采用的sgRNA序列为仓鼠LDL受体基因的互补序列,其靶向LDL受体基因为第二外显子。例如,在一个实施方案中,所采用的 CRISPR-Forward和sgRNA-Reverse序列分别如SEQ ID NO:2和3所示。
在一些实施方案中,该仓鼠为LDL受体基因敲除纯合子。在另一些实施方案中,该仓鼠为LDL受体基因敲除杂合子。
本发明另一方面提供了一种筛选用于治疗人类心血管疾病的药物的方法,包括以下步骤:
a)将候选药物给予LDL受体基因被敲除的基因工程仓鼠,
b)测定给药之前和给药之后所述基因工程仓鼠体内的血脂水平,以及
c)如果所述基因工程仓鼠体内的血脂水平在给药之后显著降低,则所述候选药物被评定为有效。
在一些实施方案中,所述心血管疾病可以选自由高脂血症、高胆固醇血症和动脉硬化症构成的组。
在一些实施方案中,所述血脂水平可以为血液胆固醇水平和/或甘油三酯水平。
在一些实施方案中,该仓鼠为LDL受体基因敲除纯合子。在另一些实施方案中,该仓鼠为LDL受体基因敲除杂合子。
本发明另一方面提供了所述基因工程仓鼠在筛选药物中的应用,其中所述药物用于治疗人类心血管疾病。
在一些实施方案中,所述心血管疾病选自由高脂血症、高胆固醇血症和动脉硬化症构成的组。
附图说明
图1显示了LDL-R基因敲除仓鼠的构建。(A)引导RNA在LDL-R基因第二外显子上识别位点示意图,其中蓝色字体为识别序列,红色字体为PAM序列。(B)两只构建成功乳鼠的LDL-R基因序列片段,其中蓝色字体为识别序列,红色字体为PAM序列,绿色字体为点突变序列,虚线部分为缺失序列,突变类型标注于每行末端。(C)LDL-R缺失Δ194bp仓鼠组织经PCR扩增后的DNA凝胶电泳图,缺失片段DNA长度为394bp。
图2显示了F1代LDL-R杂合子仓鼠血脂明显升高,血浆中LDL胆固醇比例增高。(A)LDL-R基因缺失碱基Δ10bp、Δ12bp、Δ14bp、Δ194bp的F1代杂合子及野生型对照仓鼠在普通饮食(before)和高脂饮食(2weeks)喂饲两周后的血浆总胆固醇(左图)及甘油三酯(右图)浓度。(B)缺失Δ194杂合子及野生型对照仓鼠在普通饮食(CD)及高脂饮食(HFD)喂饲两周后的血浆脂蛋白胆固醇分布。以上各组n=5,与野生型对照组相比,有显著的统计学差异(*为P0.05,**为P<0.01,***为P<0.001)。
图3显示了LDL-R基因敲除仓鼠血脂与人、小鼠的比较。(A)正常人、LDL-R杂合缺陷及纯合缺陷病人,与野生型、LDL-R杂合子及纯合子的小鼠、仓鼠的血浆胆固醇(左图)及甘油三酯(右图)浓度。(B)正常人、LDL-R杂合缺陷及纯合缺陷病人,与野生型、LDL-R杂合子及纯合子的小鼠、仓鼠的血浆脂蛋白胆固醇分布。以上各组中,人类血浆样品n=1,小鼠及仓鼠血浆样品n≥5。
图4显示了依折麦布显著逆转LDL-R基因敲除仓鼠的高脂血症。(A)LDL-R基因缺陷Δ194bp杂合子仓鼠及野生型对照仓鼠,在高脂饮食喂饲同时给予每日2mg/kg依折麦布或蒸馏水灌胃的0周、1周及2周后血浆胆固醇(左图)及甘油三酯(右图)浓度。(B)各组仓鼠在两周内的体重变化曲线,红色箭头指出禁食12小时取血的时间点。LDL-R基因缺陷Δ194bp杂合子仓鼠及野生型对照仓鼠在高脂饮食喂饲同时给予每日2mg/kg依折麦布或蒸馏水灌胃两周后的血浆脂蛋白胆固醇分布。以上各组n=5,与野生型仓鼠蒸馏水灌胃组相比,*为P<0.05,**为P<0.01,***为P<0.001;与LDL-R基因缺陷Δ194bp杂合子仓鼠蒸馏水灌胃组相比,#为P<0.05,##为P<0.01,###为P<0.001。
具体实施方式
本文所用的术语“仓鼠”是指叙利亚金黄地鼠(golden Syrian hamster)。
本文所用的术语“LDL”是指低密度脂蛋白(low density lipoprotein)。
本文所用的术语“LDL-R”是指低密度脂蛋白受体(low density lipoproteinreceptor)。在本文中,术语“LDL-R”、“LDL受体”和“低密度脂蛋白受体”可以互换使用。
本文所用的术语“KO”是指基因敲除(knock-out)。例如“LDL-R KO”是指低密度脂蛋白受体基因被敲除。
本文所用的术语“sgRNA”是指与靶基因序列互补的、引导Cas9对靶基因进行切割的RNA(single guide RNA)。
本文所用的术语“心血管疾病”泛指由于高脂血症、血液黏稠、动脉粥样硬化、高血压等所导致的心脏、大脑及全身组织发生的缺血性或出血性疾病,包括但不限于高脂血症、高胆固醇血症和动脉硬化症等。
CRISPR/Cas9基因编辑技术是本领域公知的。CRISPR(Clustered regularlyinterspaced short palindromic repeats),被称为规律成簇间隔短回文重复,实际上就是一种基因编辑器,是细菌用以保护自身对抗病毒的一个***,也是对付攻击者的基因武器。后来,研究人员发现,它似乎是一种精确的万能基因武器,可以用来删除、添加、激活或抑制其他生物体的目标基因,是一种可以广泛使用的生物技术。
CRISPR簇是一个广泛存在于细菌和古生菌基因组中的特殊DNA重复序列家族,其序列由一个前导区(Leader)、多个短而高度保守的重复序列区(Repeat)和多个间隔区(Spacer)组成。前导区一般位于CRISPR簇上游,是富含AT长度为300~500bp的区域,被认为可能是CRISPR簇的启动子序列。重复序列区长度为21~48bp,含有回文序列,可形成发卡结构。重复序列之间被长度为26~72bp的间隔区隔开。Spacer区域由俘获的外源DNA组成,类似免疫记忆,当含有同样序列的外源DNA入侵时,可被细菌机体识别,并进行剪切使之表达沉默,达到保护自身安全的目的。
通过对CRISPR簇的侧翼序列分析发现,在其附近存在一个多态性家族基因。该家族编码的蛋白质均含有可与核酸发生作用的功能域(具有核酸酶、解旋酶、整合酶和聚合酶等活性),并且与CRISPR区域共同发挥作用,因此被命名为CRISPR关 联基因(CRISPRassociated),缩写为Cas。目前发现的Cas包括Cas1~Cas10等多种类型。Cas基因与CRISPR共同进化,共同构成一个高度保守的***。
当细菌抵御噬菌体等外源DNA入侵时,在前导区的调控下,CRISPR被转录为长的RNA前体(Pre RISPR RNA,pre-crRNA),然后加工成一系列短的含有保守重复序列和间隔区的成熟crRNA,最终识别并结合到与其互补的外源DNA序列上发挥剪切作用。
目前发现的CRISPR/Cas***有三种不同类型即I型、II型和III型,它们存在于大约40%已测序的真细菌和90%已测序的古细菌中。其中II型的组成较为简单,以Cas9蛋白以及向导RNA(gRNA)为核心组成,也是目前研究中最深入的类型。
在II型***中pre-crRNA的加工由Cas家族中的Cas9单独参与。Cas9含有在氨基末端的RuvC和蛋白质中部的HNH2个独特的活性位点,在crRNA成熟和双链DNA剪切中发挥作用。此外,pre-crRNA转录的同时,与其重复序列互补的反式激活crRNA(Trans-activatingcrRNA,tracrRNA)也转录出来,并且激发Cas9和双链RNA特异性RNase III核酸酶对pre-crRNA进行加工。加工成熟后,crRNA、tracrRNA和Cas9组成复合体,识别并结合于crRNA互补的序列,然后解开DNA双链,形成R-loop,使crRNA与互补链杂交,另一条链保持游离的单链状态,然后由Cas9中的HNH活性位点剪切crRNA的互补DNA链,RuvC活性位点剪切非互补链,最终引入DNA双链断裂(DSB)。CRISPR/Cas9的剪切位点位于crRNA互补序列下游邻近的PAM区(Protospacer Adjacent Motif)的5'-GG-N18-NGG-3'特征区域中的NGG位点,而这种特征的序列在每128bp的随机DNA序列中就重复出现一次。研究结果表明,Cas9还可以剪切线性和超螺旋的质粒,其剪切效率堪比限制性内切酶。
研究概述
高胆固醇血症为心脑血管疾病的重要危险因素,并与诸多其它遗传和环境因素有相互作用。在生物医学领域研究中,用于研究高胆固醇血症疾病的动物模型,主要以低密度脂蛋白受体基因敲除(LDL-R KO)、载脂蛋白E基因敲除(ApoE KO)小鼠为主。但小鼠血脂代谢特点,心脑血管等并发症形成的机制,以及对药物的反应等方面与人类相差较大,其研究结果在临床应用方面有众多争议。而仓鼠(Golden Syrian Hamster)在糖脂代谢方面,和大鼠与小鼠相比则更接近人类。
在前期工作中,我们首创了基因工程仓鼠制备方法。在本研究中,我们应用CRISPR/CAS9***结合独创的仓鼠基因工程专利技术,敲除了仓鼠LDL-R基因。通过检测LDL-R杂合子和纯合子仓鼠血脂水平,并与人类LDL-R杂合及纯合突变的患者进行比较,发现LDLR缺陷的仓鼠模型与人类家族性高胆固醇血症高度相似,具有明显的显性遗传特征。LDL-R杂合子仓鼠胆固醇水平较野生型明显为高,而在喂饲高脂饲料(0.5%胆固醇,10%脂肪)两周时,胆固醇水平便达到2231mg/dL,是野生型仓鼠的5.5倍;给予临床上常用的降胆固醇药物依折麦布(2mg/kg)后,血浆胆固醇可降低到喂饲正常饲料的野生型仓鼠水平,脂蛋白改变以VLDL-C和LDL-C降低为主,HDL-C无明显变化。因此,与LDLR和ApoE 基因敲除的小鼠不同,LDL-R敲除的杂合子仓鼠就可以直接用于高脂血症和动脉硬化研究,是和人类LDL受体杂合子基因突变显性遗传的家族性高胆固醇血症高度相似的一个不可多得的小动物模型。
实施例1
仓鼠(叙利亚金黄地鼠,golden Syrian hamster)是一种小型啮齿类动物,广泛用于各个研究领域,包括肿瘤、病毒学、糖尿病及心血管疾病等(7-10)。在糖脂代谢方面,仓鼠具有小鼠大鼠所不具备的、但与人类相似的特征:胆固醇酯转运蛋白(cholesteryl estertransfer protein,CETP)高表达(11),只有小肠才有载脂蛋白B的编辑酶活性以及高脂饲料诱发的复合型高脂血脂(12,13)。因此,仓鼠很有可能成为最理想的高脂血症小动物模型,用于高脂血症及其并发症发病机制和药物疗效研究。
我们近期在仓鼠胚胎操作的技术上有了重大的突破,成功制备了国际上第一例基因工程仓鼠(14)。在此基础上,本研究利用CRISPR/CAS9基因编辑技术(15,16),构建了LDL-R基因敲除的仓鼠,并对其表型与LDL-R基因突变的家族型高胆固醇血症患者以及LDL-R KO小鼠进行了比较分析。
实验方法和材料
1.实验动物的种类及饲养
本项目使用的野生型仓鼠来自北京维通利华实验动物技术公司,按清洁级标准饲养。保持温度24℃,湿度50-60%,光照周期为6:00-20:00光照、20:00-6:00黑暗。基因敲除仓鼠模型的所有实验操作过程均通过北京大学实验动物伦理委员会审查。
2.CRISPR/Cas9***构建
2.1仓鼠LDL受体基因特异性打靶序列的设计
仓鼠LDL受体基因特异性打靶DNA序列的识别是根据CRISPRE-Cas9打靶的原则,在叙利亚金黄地鼠LDL受体基因的外显子中,选择GG(N)18NGG的序列,应用用BLAST(http://blast.ncbi.nlm.nih.gov/Blast.cgi)方法,在叙利亚金黄地鼠的全基因组序列中,进行特异性验证。若发现其它基因组序列与该GG(N)18NGG序列的最后12位碱基以及PAM序列完全匹配,则认为该序列有潜在的脱靶效应,即舍弃该打靶序列,重新进行设计。
根据上述原则,我们采用仓鼠ldlr基因中的打靶序列为gaaatgcatcgccagcaag(SEQ ID NO:1)。
2.2Cas9mRNA的制备
本项目采用的cas9DNA模板是含有人源化cas9cDNA的PXT7质粒(17)。 PXT7-cas9具有氨苄青霉素抗性,可用于质粒扩增;XbaI酶切位点,可用于质粒线性化。
转录前,PXT7-cas9在XbaI的作用下充分线性化。反应终止后,蛋白酶K处理0.5小时,尽可能除去样品中的RNA酶。进一步酚-氯仿抽提,乙醇沉淀,得到可用于转录的DNA模板。
体外转录试剂盒为mMESSAGE mMACHINE T7kit(Ambion)。转录体系于37℃充分反应2小时。反应所得cas9mRNA用酚-氯仿抽提的方法进行纯化。异丙醇沉淀后,用无RNA酶水溶解,使其浓度为500ng/μl。琼脂糖凝胶电泳检测所得mRNA的大小,评估其降解情况。分装后,储存于-80℃,用于注射。
2.3sgRNA(single-guide RNA)的制备
sgRNA模板通过下述两条合成的有互补序列的引物通过互为模板PCR方式扩增得到(18)。分别合成的两条序列:1)特定的寡核苷酸序列CRISPRF,该序列包含T7启动子,sgRNA序列和部分sgRNA骨架部分;2)共同的序列sgRNA,即骨架部分。
CRISPR-Forward=GAAATTAATACGACTCACTATAGGAAATGCATCGCCAGCAAGGTTTTAGAGCTAGAAATAGC(SEQ ID NO:2)
sgRNA-Reverse=AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC(SEQ ID NO:3)
PCR于50μl体系中进行,反应条件为(98℃30s,35cycles of[98℃10s,60℃30s,72℃15s],72℃10min,4℃∞)。PCR产物经过琼脂糖凝胶电泳,用胶回收试剂盒(TAKARA)回收120bp的DNA条带,即sgRNA的模板DNA。蛋白酶K处理0.5小时,尽可能除去样品中的RNA酶。进一步,酚仿抽提,乙醇沉淀,得到可用于转录的DNA模板。
体外转录试剂盒为Megascript T7Kit(Ambion),转录体系于37℃充分反应4小时。反应所得sgRNA mRNA用MEGAclear Kit(ambion)进行纯化。无RNase水溶解使其浓度为200ng/μl。琼脂糖凝胶电泳检测所得mRNA的大小,评估其降解情况。分装后,储存于-80℃,用于注射。
3.基因敲除仓鼠构建
3.1仓鼠受精卵的收集和培养
选取8-12周龄雌鼠诱导其超数***。仓鼠动情周期为四天,在周期第二天11:00,即发情症状消失后,腹腔注射20IU孕马血清***(PMSG)。82小时后,即下一周期的发情期,与雄鼠合笼进行交配。PMSG注射98小时后,雌鼠腹腔注射过量的戊巴比妥钠处死。将其输卵管剪下,置于37℃预热的M2培养基中(Sigma-Aldrich,St.Louis,MO,USA)。在解剖显微镜下,将输卵管膨大的壶腹部撕破,含有受精卵的堆积物流出,收集受精卵。
仓鼠受精卵的体外培养基是HECM-10(NaCl 113.8mM,KCl 3mM,NaHCO3 25 mM,sodium lactate 4.5mM,CaCl2 1mM,MgCl2 2mM,glutamate 0.01mM,glutamine 0.2mM,glycine 0.01mM,histidine 0.01mM,lysine 0.01mM,proline 0.01mM,serine 0.01mM,asparagine 0.01mM,aspartate 0.01mM,cysteine 0.01mM,taurine0.5mM,pantothenate0.003mM,PVA0.1mg/ml,Sigma-Aldrich)(19)。用石蜡油将HECM-10培养液滴覆盖。受精卵培养温度为37.5℃,CO2浓度为10%(14)。
3.2显微注射
制备注射液滴,注射皿中,加入一滴100μl M2培养基,矿物油液封。所有的受精卵离开孵箱时间少于15分钟(20)。sgRNA和cas9mRNA共同注射至细胞质中,其注射浓度分别为20ng/μl和50ng/μl。注射后受精卵在孵箱中培养0.5小时后,用于回输***母鼠体内。
3.3受精卵植入
挑选与供卵仓鼠年龄相同、动情周期一致的雌鼠作为***仓鼠。***鼠、供卵鼠同步,与雄鼠交配。次日,将注射过的受精卵从输卵管伞口植入***鼠体内。每侧输卵管植入15个受精卵。
3.4基因型分析
出生仓鼠一周龄后,取其脚趾组织进行DNA提取鉴定。将突变位点进行PCR扩增(ldlr-F=CGGCCCAGATGTCAATAT(SEQ ID NO:4),ldlr-R=GTGAAACCCTCCAAACCC(SEQ IDNO:5)),所得产物测序鉴定。发生突变的仓鼠,进一步序列验证:将其PCR产物连接至pEASY-T1载体中(transgen biotech),转化培养后,挑取部分单克隆进行测序。
4.高脂饲料喂饲及依折麦布实验性治疗
本研究采用的高脂饲料为含0.5%胆固醇,10%猪油的普通啮齿类饲料。依折麦布的给药剂量为2mg/kg体重,灌胃给予,对照组给予三蒸水。分别于高脂前、高脂后两周,给药前、给药后两周采集血浆。
仓鼠经过夜禁食12小时,由腹腔注射戊巴比妥钠麻醉后,经眼眶后静脉采血约1ml,肝素抗凝。
5.血浆脂质检测
血浆甘油三酯测定试剂盒、总胆固醇测定试剂盒(中生北控生物科技有限公司)用于检测血浆甘油三酯、总胆固醇水平。高密度脂蛋白胆固醇(HDL-C)采用聚乙二醇沉淀法去除含apoB的脂蛋白后,检测其胆固醇水平。
血浆脂蛋白中脂质组分检测,采用美国Amersham Biocsciences公司的快速蛋白液相色谱(fast protein liquid chromatography,FPLC)仪,superose 6HR10/30色谱柱首先分离血浆脂蛋白组分。血浆200μl通过0.22μm针筒式过滤器后上样,以每分钟0.5mL的速度自动收集分离组分,每个组分500μl,共35个组分。对各个组分应用上述胆固醇检测试剂盒检测胆固醇含量,做出血浆脂蛋白谱。
结果
LDL-R基因突变仓鼠的构建。我们以LDL-R基因第二外显子为靶点(图1A),设计特异性识别LDL-R基因的引导RNA。120个受精卵经细胞质注射后,回输至***4只母鼠体内。共出生16只幼崽,经PCR后测序鉴定,其中两只为LDL-R缺陷仓鼠。cas9内切酶在特异性位点酶切后,受损DNA由末端直接连接或同源重组进行修复,因此产生体内有多种不同基因型的LDL-R基因突变嵌合体仓鼠(founders,图1B)。其中,founder1、fouder2分别是含有四种不同基因型、194bp大片段缺失和点突变的嵌合体。图1C显示了founder2的F2代基因型鉴定的琼脂糖凝胶电泳结果。由此建立的LDL-R基因敲除仓鼠均可生育。
LDL-R基因缺失导致不同程度的血脂升高。我们对不同LDL-R基因缺失的founders以及F1代杂合子和F2代纯合子仓鼠的表型进行了分析。
本课题所构建的founder1、fouder2分别是含有四种不同基因型、194bp大片段缺失和点突变的嵌合体。12周时,血浆胆固醇水平分别为578mg/dL,429mg/dL,同窝野生型对照为189mg/dL和161mg/dL。甘油三酯水平达到422mg/dL和215mg/dL,显著高于其同窝WT的甘油三酯水平99mg/dL和137mg/dL(图2A)。
Founders与WT仓鼠交配,产生6种基因型的杂合子LDL-R基因突变仓鼠。在普通饲料喂饲下,LDL-R杂合型(+/-)Δ194bp(194个碱基对缺失)、Δ10bp(10个碱基对缺失)、Δ14bp(14个碱基对缺失)平显著高于野生型(wild type,WT)。在高脂饲料喂饲两周后,LDL-R+/-Δ194bp和Δ10bp胆固醇水平最高达到2231mg/dL,与高脂前相比升高了7.6倍,是高脂WT的5.5倍。LDL-R+/-Δ12bp、和Δ14bp胆固醇水平分别达到881mg/dL、645mg/dL,分别是WT的2.2和1.6倍(图2B)。不同的LDL-R+/-基因型之间胆固醇水平存在一定差异。图2B显示普通饲料时各个基因型LDL-R+/-的甘油三酯水平为190mg/dL,与WT没有显著差异。高脂两周后,LDL-R+/-Δ194bp、Δ10bp甘油三酯水平最高达到1040mg/dL,与高脂前相比升高了4.4倍,是高脂WT的4.1倍。与胆固醇变化趋势类似,LDL-R+/-Δ12bp、和Δ14bp高脂饲料后甘油三酯水平分别达到570mg/dL、310mg/dL,分别是WT的2.28和1.24倍。以下研究主要以基因型为LDL-R+/-Δ194bp和Δ10bp的仓鼠为主进行分析。
血浆脂蛋白FPLC分析的结果显示(图2C),在普通饲料喂饲下,LDL-R+/-Δ194bp仓鼠与WT比较,LDL含量显著增高,而HDL水平没有改变。提示仓鼠在LDL-R基因缺失一半的情况下就有LDL-C升高的表型,这与临床家族性高胆固醇血症的显性遗传特征类似,即杂合子便有明显的高胆固醇血症表型。高脂饲料喂饲两周后,WT血浆脂质中VLDL和LDL均有显著升高,LDL-R+/-血浆脂质以VLDL、LDL升高为主,HDL水平没有明显变化。
LDL-R基因敲除仓鼠血脂与人、小鼠的比较。为了进一步确定LDL受体功能缺失,我们对F2代仓鼠野生型、杂合子、纯合子血脂情况进行检测。LDL-R敲除纯合子(LDL-R-/-)血浆总胆固醇水平达到833mg/dL,是其同窝对照野生型的6.4倍。LDL-R基因敲除杂合子(LDL-R+/-)血浆胆固醇水平达到237.8mg/dL,约为野生型1.8倍(图3A)。为了与LDL-R-/-仓鼠作比较,我们分别收集了一例家族型 高胆固醇血症杂合子(26岁,男)和纯合子患者(8岁,男,服用10mg他汀)血浆。与小鼠LDL-R基因缺陷相比,LDL-R-/-仓鼠具有更接近家族性高胆固醇血症患者的胆固醇水平。与小鼠和人LDL-R缺陷不同,LDL-R-/-仓鼠甘油三酯水平达到350mg/dL,是同窝野生型对照仓鼠的1.7倍(图3B)。FPLC的结果显示,与人类不同,小鼠血浆胆固醇主要载体为HDL,有少量的LDL,几乎没有VLDL。LDL-R+/-中,脂质组分仍以HDL为主,LDL几乎没有改变(21)。仅在纯合子LDL-R基因敲除小鼠,LDL水平才有所升高。而野生型仓鼠血浆中就有一定含量的LDL,而在LDL-R+/-仓鼠中,血浆LDL明显升高,VLDL和HDL没有改变。而纯合子LDL-R基因敲除仓鼠的VLDL与LDL均显著升高。
依折麦布显著逆转LDL-R基因敲除仓鼠的高脂血症。和小鼠相比,仓鼠在血浆脂质和脂蛋白组成与人类更加接近。为了验证临床上常用的降脂药物是否对仓鼠同样具有治疗作用,我们检测了LDL-R+/-仓鼠给予高脂饲料后再给予依折麦布,观察血浆脂质和脂蛋白的改变。图4A显示,高脂饲料同时给予依折麦布2mg/kg灌胃治疗两周后,单纯高脂WT和LDL-R+/-仓鼠的血浆胆固醇水平为513mg/dL、1864mg/dL,给药后分别降至111mg/dL和157mg/dL,接近普通饲料的水平。甘油三酯水平也分别由413mg/dL、658mg/dL降至157mg/dL和225mg/dL。依折麦布给药组的体重与高脂组没有显著差异。在高脂给药7天和14天时,由于禁食取血,各个组的体重有明显下降,并在第二天恢复(图4B)。FPLC结果显示,依折麦布主要降低血浆中的VLDL和LDL,对HDL的影响不大。
讨论
本研究利用近年来最新建立基因编辑方法CRISPR/CAS9***,成功构建了LDL-R基因敲除仓鼠。在传统的基因敲除技术中,采用同源重组的策略,然而这种方法效率较低,只能在胚胎干细胞上进行操作,限制了其在小鼠之外其他物种上的应用。随着CRISPR/CAS9、TALENs等高效基因编辑方法的建立完善,基因打靶技术产生重大变革,其效率大大提高,在受精卵的水平进行操作便可高效得到基因修饰的品系,大大扩展了基因修饰的物种(22)。与小鼠大鼠相比,仓鼠胚胎对体外操作极为敏感。在前期工作中,我们在仓鼠胚胎操作技术上有了重大的突破,在国际上首先报道了基因工程仓鼠的成功制备(14)。仓鼠在血浆脂蛋白代谢及免疫反应方面具有其他啮齿类动物不具备的特征,而与人类更接近。因此,基因工程仓鼠的诞生为人类疾病研究提供了更为理想的动物模型。
在LDL-R基因敲除仓鼠的构建中,由于cas9内切酶在识别特异性位点酶切后,受损DNA随机由末端直接连接或同源重组进行修复,因此产生LDL-R基因不同程度突变仓鼠。我们不仅得到了194bp大片段缺失的LDL-R基因敲除仓鼠,而且得到点突变和4氨基酸缺失的LDL-R基因缺陷的动物模型。在初步的血脂分析中,不同类型的LDL-R+/-仓鼠与野生型相比,胆固醇水平均有不同程度的升高。这与高胆固醇血症患者LDL-R基因多态性相似,可以用于不同程度LDL-R基因缺陷病人发病机制的研究,并提供不同的临床治疗策略。
现已知的LDL-R基因突变,发生在渡边家兔,猕猴,LDL-R敲除小鼠以及人类中的家族型高胆固醇血症患者中(23,24)。在LDL-R敲除小鼠诞生之前,主要用于高脂血症和动脉粥样硬化研究的模型为渡边家兔。与目前最常用的LDL-R敲除小鼠相比,仓鼠和家兔具有类人的脂代谢特征:CETP高表达,对饮食诱导的代谢综合征敏感,只有小肠才有载脂蛋白B的编辑酶活性(25)。与高胆固醇血症患者相似,渡边家兔在12月龄时,血浆胆固醇升至700–1200mg/dL的极高水平,而LDL-R敲除小鼠仅在高脂饮食诱导下,才会引起血浆胆固醇极度升高。本研究构建的LDL-R敲除仓鼠纯合子在6周时血浆胆固醇可达到833mg/dL。与人类高胆固醇血症患者相似,LDL-R基因突变仓鼠亦为显性遗传,即LDL-R+/-仓鼠具有高脂血症的表型。这些是LDL-R和ApoE基因敲除小鼠所不具备的特征。这意味着,LDL-R敲除杂合子仓鼠可以直接用于研究,且代表了临床上数量庞大的杂合子家族性高胆固醇血症的患者(以1/200计,中国存在6-7百万患者,全世界则可能有3千多万)。由于LDL-R基因突变为显性遗传,因此LDL-R+/-仓鼠在具有高脂血症表型的同时,仍有一半的LDL-R的发挥作用,可以应用于新型药物,如前蛋白转化酶枯草杆菌蛋白酶Kexin-9型(PCSK9)抑制剂等影响LDL-R功能实验性研究。
此外,通过选育后,部分严重高胆固醇血症渡边家兔可发生严重的冠脉病变和心肌梗死。但家兔的缺陷在于其致病脂蛋白为β-VLDL,不经氧化修饰就可被巨噬细胞大量吞噬;缺乏肝脂酶、ApoE和ApoAII;而且作为草食动物对胆固醇毒性作用敏感,所以动脉粥样硬化的发生机制不同于人类。更由于渡边家兔繁育困难,数量有限,因此在几十年中,并没有得到在高胆固醇血症和动脉粥样硬化研究领域中的广泛应用。而小鼠动脉粥样病变多发于主动脉、流出道,极少累及冠脉和脑动脉。基于小鼠高胆固醇血症的心脑血管并发症的研究,缺乏冠状动脉和脑动脉病变的基础病因和病生理基础,不能模拟人类冠心病和中风的自然发病过程,有明显局限性。有文献报道(26,27),仓鼠在高胆固醇饮食10个月时,出现冠脉动脉粥样硬化病变。这提示,仓鼠是冠状动脉粥样硬化易感的物种。在LDL-R基因敲除的仓鼠中,很有可能加速病程的进展,加重疾病的发生,从而导致类似人类冠心病自然发病过程。因此,仓鼠不仅是很好的高胆固醇血症小动物模型,而且有可能是一个与人类疾病发病机制更为接近的动脉粥样硬化以及冠心病和中风的模式动物。
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Claims (10)
1.制备基因工程仓鼠的方法,包括:将该仓鼠体内的LDL受体基因敲除。
2.权利要求1的方法,其中所述LDL受体基因通过CRISPR/CAS9基因编辑技术被敲除。
3.权利要求1或2的方法,其中所采用的sgRNA序列为仓鼠LDL受体基因的互补序列,其靶向LDL受体基因为第二外显子。
4.权利要求1或2的方法,其中该仓鼠为LDL受体基因敲除杂合子。
5.筛选用于治疗人类心血管疾病的药物的方法,包括以下步骤:
a)将候选药物给予LDL受体基因被敲除的基因工程仓鼠,
b)测定给药之前和给药之后所述基因工程仓鼠体内的血脂水平,以及
c)如果所述基因工程仓鼠体内的血脂水平在给药之后显著降低,则所述候选药物被评定为有效。
6.权利要求5的方法,其中所述心血管疾病选自由高脂血症、高胆固醇血症和动脉硬化症构成的组。
7.权利要求5或6的方法,其中所述血脂水平为血液胆固醇水平和/或甘油三酯水平。
8.权利要求5或6的方法,其中所述基因工程仓鼠为LDL受体基因敲除杂合子。
9.权利要求1-4之任一项的方法所制备的基因工程仓鼠在筛选药物中的应用,其中所述药物用于治疗人类心血管疾病。
10.权利要求9的应用,其中所述心血管疾病选自由高脂血症、高胆固醇血症和动脉硬化症构成的组。
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CN108753837A (zh) * | 2018-06-15 | 2018-11-06 | 扬州大学 | 一种高血脂或动脉粥样硬化兔模型的构建方法及sgRNA |
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