CN111607597A - Asgr1突变基因在制备拟人化的低血脂代谢动物模型中的应用 - Google Patents
Asgr1突变基因在制备拟人化的低血脂代谢动物模型中的应用 Download PDFInfo
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Abstract
本申请属于基因工程技术领域,公开了ASGR1突变基因在制备拟人化的低血脂代谢动物模型中的应用。通过对ASGR1基因序列的合适的编辑位点进行基因的编辑,获得的动物对高脂高糖饮食(动脉粥样硬化的主要诱因)有明显耐受作用,可做为预防人类动脉粥样硬化疾病的模型;可自然繁育并建立了稳定的繁育群体,可满足该基因突变模型规模化的制备需求。本发明还建立了一个可规模化制备拟人化低血脂代谢动物模型的方法。
Description
技术领域
本发明属于基因工程技术领域,具体涉及ASGR1突变基因在制备拟人化的低血脂代谢动物模型中的应用。
背景技术
动物疾病模型是指各种医学科学研究中建立的具有人类疾病模拟表现的动物。主要用于实验生理学、实验病理学和实验治疗学(包括新药筛选)研究。人类疾病的发展十分复杂,以人本身作为实验对象来深入探讨疾病发生机制,推动医药学的发展来之缓慢,临床积累的经验不仅在时间和空间上都存在局限性,而且许多实验在道义上和方法上也受到限制。而借助于动物模型的间接研究,可以有意识地改变那些在自然条件下不可能或不易排除的因素,以便更准确地观察模型的实验结果并与人类疾病进行比较研究,有助于更方便、更有效地认识人类疾病的发生发展规律,研究防治措施。
心血管疾病是心脏或者血管病变引发的疾病,是全球死亡率最高的疾病。动脉粥样硬化(Atherosclerosis,As)是其发病的主要根源之一。目前,全球每年因心血管疾病死亡人数已超过1.7亿,占总死亡人数的31%,是致死率最高的疾病。虽然高脂高糖饮食被广泛认为是动脉粥样硬化的主要诱因,但动脉粥样硬化的致病因素众多,发病机制复杂,其发病机理目前尚未被完全阐明。因此,建立动物模型研究并阐明动脉粥样硬化的发病机理,对预防和治疗心血管疾病具有重要的临床应用价值和意义。
血脂代谢异常主要和遗传及饮食环境有关。目前血脂代谢模型主要集中于高血脂症动物模型,低血脂症模型的制备尚无有效实现方案。
制备动物模型一般方法有外科手术、基因工程、药物诱导等方法。然而a)外科手术尚无有效的术式可造成低血脂症状;b)基因工程制备低血脂模型理论上可通过对LDLR、PCSK9、LDL-c等基因靶点的编辑实现,现在尚无成功的模型问世;c)与上述相关的基因的抗体理论上可以用作药物来饲喂动物产生低血脂模型,尚无实际案例。
2016年,Paul Nioi等人通过大规模测序发现:冰岛地区人群中ASGR1基因的突变与心血管疾病的发病率呈强负相关,携带有该基因内含子12bp缺失的人群(1/120),其血液中非HDL-c水平显著低于对照组。然而,研究表明ASGR1缺失小鼠(无论是在正常遗传背景下还是在LDLR缺失的背景下)血液中LDL-c水平与对照组小鼠相似(Ryuichi et al.2001)。以小鼠为模型建立拟人化的低血脂代谢动物模型的研究结果与人类ASGR1基因突变表型相矛盾。
总之,基因工程与药物诱导是最可能实现低血脂代谢的模型技术,然而基因工程动物制备成本高、较一般动物更难以存活,无法形成有效的繁育群体;药物诱导则存在有效验证、抗体费用高、给药效果因动物个体差异存在表型不一致等原因,限制了其发展。
发明内容
有鉴于此,本发明的目的在于针对现有技术的缺陷,提供一种基因工程编辑位点,用于制备低血脂代谢动物模型。
申请人在大动物ASGR1基因合适位点进行基因的编辑,获得了对高脂高糖饮食有明显耐受作用的大动物模型。因此本发明提供了所述ASGR1突变基因大动物的制备方法及其在拟人化的低血脂代谢动物模型中的应用。
其中,所述ASGR1突变基因为ASGR1基因第2817~2961位发生突变。
突变是指生物体基因组核苷酸序列的改变。本发明所述突变包括一个或多个核苷酸的插入、缺少、替换。
在本发明所述多个为2个、3个、4个、……146个、147个……156个。
在本发明一些实施方案中,所述ASGR1突变基因为ASGR1基因第5外显子上的两个等位基因第2840~2859位均发生20bp删除,命名为(-20bp/-20bp)突变型ASGR1突变基因。
在本发明另一些实施方案中,所述ASGR1突变基因为ASGR1基因第5外显子上的一个等位基因第2817~2961位发生146bp删除,另一个等位基因第2852~2853位增加1bp,命名为(-146bp/+1bp)突变型ASGR1突变基因。
在本发明中,所述动物为猪。在一些实施方案中,所述动物为巴马小型猪。
在本发明中,所述拟人化的低血脂代谢为与人ASGR1基因突变产生的低血脂表型一致。
进一步的,本发明还提供了拟人化的低血脂代谢动物模型的制备方法,采用基因编辑技术制备具有ASGR1突变基因的动物模型。
在一些实施方案中,所述制备方法为采用基因编辑技术制备具有所述(-20bp/-20bp)突变型或(-146bp/+1bp)突变型的ASGR1突变基因的动物模型。
在一些实施方案中,所述基因编辑技术为CRISPR/Cas9基因编辑技术。本领域技术人员可以理解本发明所述制备方法并不限于采用第三代基因编辑技术crisper/cas9技术实现猪的基因组编辑,其它的基因组编辑技术也可以实现相同效果,包括但不限于锌指核酸内切酶(Zinc figer nucleases,ZFNs)定向修饰靶基因编辑技术、类转录激活因子效应物核酸酶(transcription activator-like effector nuclease,TALENs)定向修饰靶基因编辑技术。
进一步的,在一些实施方案中,所述的制备方法利用CRISPR/Cas9基因编辑技术制备所述ASGR1突变基因的动物模型的方法具体为在ASGR1基因第5外显子设计并合成特异识别靶序列DNA的sgRNA,构建含有sgRNA的表达载体用于细胞转染受体细胞,核移植制备动物胚胎,移植到发情的受体动物子宫内制备。
在一些实施例中,所述sgRNA的序列如SEQ ID NO:1所示,以制备获得ASGR1突变基因的动物模型。
所述的合成的sgRNA寡聚核苷酸退火后连接表达载体。将构建好的表达载体测序验证连接正确后,提取质粒用于细胞转染。其中所述退火为94℃,10min;37℃,10min。
在一些实施例中,所述表达载体为PX330表达载体。
在本发明中,将猪耳成纤维细胞复苏,用含sgRNA-ASGR1敲除质粒的电转液重悬后采用Lonza核转染仪按T-16程序进行电转染。并采用流式筛选可用于移植的克隆点。选取ASGR1敲除的克隆点用于核移植制备基因编辑动物胚胎并将其移植到发情的受体母猪子宫内获得ASGR1基因敲除猪。
由上述技术方案可知,本发明提供了ASGR1突变基因在制备拟人化的低血脂代谢动物模型中的应用。通过对ASGR1基因的编辑获得的动物对高脂高糖饮食(动脉粥样硬化的主要诱因)有明显耐受作用,且可自然繁育、建立了稳定的群体,可满足规模化的制备需求,做为预防人类动脉粥样硬化疾病模型,进一步研究ASGR1突变人群低血脂抗动脉粥样硬化的机制及相关的药物毒性研究。本发明还建立了一个可高效建立可自然繁育并对高脂高糖饮食有明显耐受作用拟人化的低血脂代谢动物模型的制备方法,可用于拟人化的低血脂代谢动物模型的规模化制备。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示实施例1 ASGR1基因sgRNA靶点设计示意图;
图2示实施例1 ASGR1基因敲除载体设计示意图;
图3示实施例1 ASGR1基因敲除猪基因型的鉴定;其中A为获得的ASGR1基因敲除巴马小型猪;B为PCR扩增鉴定获得的Founder猪基因型,在sgRNA识别切割靶点上下游共计目的片段为676bp的鉴定引物(上游引物为AgF:5′-gagagagaccttcagcaacctc-3′;下游引物为:AgR:5′-catagtccacccagttaaccgg-3′);C为基因测序鉴定获得的Founder猪基因型;D为Westernblot鉴定最终存活的四头Founder猪耳组织中ASGR1蛋白的表达图;
图4示实施例217月龄ASGR1敲除的猪(ASGR1-KO(ASGR1-/-)和ASGR1-SKO(ASGR1+/-))与对照组血脂表型检测结果;
图5示实施例3正常饲料饲喂后苏丹IV染色血管图;
图6示实施例3高脂高糖饲喂后苏丹IV染色血管图;
图7示实施例3高脂高糖饲喂后血管内壁的脂肪颗粒组织切片HE染色及EVG染色结果图;
图8示实施例3高脂高糖饲喂后CT造影显示结果;CT造影结果显示,高脂高糖饲喂后,WT型动脉内出现阴影,可能存在病变,箭头所指为疑似阴影部位;
图9示实施例4ASGR1基因敲除仔猪系谱图;其中F0:原代猪;F1:F2代杂合子猪。
具体实施方式
本发明公开了ASGR1突变基因在制备拟人化的低血脂代谢动物模型中的应用。本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及产品已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法进行改动或适当变更与组合,来实现和应用本发明技术。
ASGR1基因与心血管疾病相关,敲除该基因的动物对高脂高糖饮食有明显耐受作用表型。本发明发现并建立了一个可高效建立可自然繁育并对高脂高糖饮食有明显耐受作用拟人化的低血脂代谢动物模型的制备方法,可用于拟人化的低血脂代谢动物模型的规模化制备。通过本发明所述制备方法获得的大动物血脂代谢模型表型均一稳定,获得原代动物后可通过自然繁育的方式进行繁殖,生产成本及规模可大幅度优化。
本发明所述拟人化的低血脂代谢大动物的制备方法产生的动物有3个特征:1)模拟人ASGR1基因突变,产生低血脂表型与人一致;2)可模拟突变人群,对动脉粥样硬化有耐受作用;3)制备的动物可传代,可用于模拟ASGR1基因突变的人群进行低血脂代谢、抗动脉粥样硬化机制、及针对ASGR1基因突变人群的药物毒性方面的研究。
为了进一步理解本发明,下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅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造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。
如无特殊说明,本发明实施例中所涉及的试剂均为市售产品,均可以通过商业渠道购买获得。
实施例1、ASGR1基因敲出仔猪的制备
根据猪(Sus scrofa)源去唾液酸糖蛋白受体1(asialoglycoprotein receptor,ASGR1)基因序列(Gene ID:NC_010454.4),利用单链导向RNA(single guide RNA,sgRNA)在线设计网站(http://crispr.mit.edu/)在其第5外显子(图1)设计并合成了特异识别靶序列DNA的sgRNA(sgRNA:agcagtttgtgtccgacctgcgg)。将合成的sgRNA寡聚核苷酸退火(94℃,10min;37℃,10min)后,连入BbsⅠ酶切回收的PX330表达载体,构建sgRNA表达载体(图2)。构建好的表达载体测序验证连接正确后,提取质粒用于细胞转染。
将验证有效的sgRNA表达载体与增强绿色荧光蛋白(Enhanced GreenFluorescent Protein,EGFP)质粒共同电转染巴马小型猪耳成纤维细胞,细胞传代培养后采用流式细胞仪富集带绿色荧光细胞,利用有限稀释的方式将富集得到的细胞稀释培养至100mm培养皿内(30cell/皿),经过15d左右培养,共获得20个单细胞克隆,其中双等位基因敲除单细胞克隆15个(75%);单等位基因敲除单细胞克隆3个(15%);野生型单细胞克隆2个(10%)(表2)。
表1阳性单细胞克隆ASGR1基因突变效率
选择不同突变类型(-20bp/-20bp,-146bp/+1bp)的ASGR1基因敲除单细胞克隆为供体细胞进行核移植,共构建克隆胚胎361枚,移植受体母猪2头,受体均妊娠到终期,分娩存活仔猪7头,如表2及图3A。
表2 ASGR1基因敲除胚胎移植后的体内发育和出生结果
注:“+”表示妊娠。
仔猪出生后,采耳组织样品提取DNA,通过PCR及测序检测ASGR1基因突变类型。在sgRNA识别切割靶点上下游共计目的片段为676bp的鉴定引物(上游引物为AgF:5′-gagagagaccttcagcaacctc-3′;下游引物为:AgR:5′-catagtccacccagttaaccgg-3′),通过westernblot检测蛋白表达(ASGR1兔多克隆抗体)。
经PCR及测序鉴定7头全部为ASGR1基因敲除猪(图3B-3C),其中3头(281、330、332)敲除仔猪在ASGR1基因的两个等位基因均发生-20bp删除,4头(331、333、334、335)基因型为(-146bp/+1bp)。Western blot验证最终存活下来的4头(330、331、332、334)ASGR1基因敲除猪肝脏组织未检测到ASGR1蛋白表达(图3D),表明利用CRISPR/Cas9成功构建了ASGR1基因敲除猪(ASGR1-KO猪)。
实施例2、ASGR1基因敲除动物模型低血脂表型与ASGR1突变人群相似性比较
对实施例1获得的ASGR1基因敲除猪进行血脂指标检测。
1、试验动物
实验组1:ASGR1-KO 3头(实施例1制备获得)
实验组2:ASGR1-SKO猪(ASGR1-KO猪与WT母猪配种繁育产生的个体)
对照组:以未进行基因编辑的野生型猪(wide type,WT)作为对照.
实验动物均在相同实验条件下饲喂.
2、试验方法:
分别在同月龄(17月龄)时对三组动物空腹采血,分离血清,检测血脂指标(血液总胆固醇TC,甘油三酯TG,LDL-c,HDL-c,Non-HDL-c,apoA1,apoB等)
3、试验结果:
对ASGR1敲除的猪(ASGR1-KO(ASGR1-/-)和ASGR1-SKO(ASGR1+/-))及对照(WT)进行血脂指标检测的统计结果见图4。
结果显示,本发明采用基因编技术,在大动物体内模拟人对ASGR1基因进行敲除,成功获得了拟人化的低血脂代谢模型,血液中非HDL-c水平显著低于对照组。ASGR1敲除的猪血液中血脂指标中Non-HDL-c、LDL-c水平显著低于对照组,与ASGR1基因突变人群表型一致。
实施例3、ASGR1基因敲除动物模型对高脂高糖诱导动脉粥样硬化的耐受
本发明对ASGR1敲除的猪与对照组进行了动脉粥样硬化的耐受实验。
采用高脂高胆固醇日粮(20%的脂肪、2%的胆固醇)诱导ASGR1敲除的猪与对照组进行动脉粥样硬化建模同时设立饲喂正常饲料的空白对照组,以期加快实验进程。采用高脂高胆固醇日粮对ASGR1敲除的猪进行为期6个月的饲养,高脂高胆固醇日粮及正常饲料配方见表3。
表3试验猪饲料配方(%)
原料名称 | 正常饲料 | 高脂高胆固醇饲料 |
玉米 | 80.2 | 61.8 |
豆粕 | 12 | 9.2 |
稻米糠 | 6 | 4.7 |
鱼粉 | 0.3 | 0.3 |
石粉 | 0.72 | 0.7 |
食盐 | 0.28 | 0.3 |
牛油 | 0 | 15 |
花生油 | 0 | 5 |
胆固醇 | 0 | 2 |
胆盐 | 0 | 0.5 |
预混料 | 0.5 | 0.5 |
合计 | 100 | 100 |
其中,预混料含有0.05%的维生素、0.25%的矿物质、0.05%的胆碱及0.05%的植酸酶;每kg预混料包括:维生素A 67IU,维生素D 16.2IU,维生素E 7.4g,维生素K 340mg,维生素B1 670mg,维生素B2 1000mg,维生素B6800mg,维生素B12 1.4mg,维生素C 10g,泛酸2.65g,叶酸330mg。
饲喂完成后,采集主动脉血管作病理检测,结果如下:
1)正常饲料饲喂,6个月后WT与ASGR1基因敲除猪主动脉血管解剖无异常,苏丹IV染色血管(苏丹IV可对脂肪着色,显示红色)未发现斑块沉积(图5)。
2)高脂高糖饲喂,6个月后,WT与ASGR1基因敲除猪主动脉血管解剖发现血管内壁有颗粒沉积,苏丹IV染色血管发现WT出现大面积红色,出现显著的斑块沉积;与ASGR1基因敲除猪相比,其着色面积(斑块沉积面积)、着色强度(斑块沉积强度)均有显著差异,动脉粥样硬化程度显著高于ASGR1基因敲除猪(图6)。
对血管内壁的脂肪颗粒进行组织切片HE染色及EVG(Verhoeffs Van Gieson染色,可对弹性纤维着黑色、胶原纤维着红色)染色。HE染色发现沉积物附着在血管表层,发现WT血管颗粒沉积的部位,弹性纤维明显减少,显示血管出现硬化病变,而ASGR1基因敲除猪血管内壁弹性纤维与胶原纤维同时存在,血管表型正常(图7)。
对高质高胆固醇饲喂组进行像学(CT)检测动脉粥样硬化斑块,结果见图8。结果显示,在野生对照组中检测到了斑块沉积、而ASGR1基因敲除猪体内未见明显异常。
上述结果表明,ASGR1基因敲除猪可耐受高脂高糖诱导动脉粥样硬化,表型与ASGR1突变人群一致。
实施例4、ASGR1基因敲除仔猪可正常传代。
以ASGR1敲除猪为例,我们通过基因编辑的方法,获得了6头原代个体。选取原代中的332与334号ASGR1-/-猪与野生型母猪交配,获得了10头F1代ASGR1+/-猪,建立ASGR1基因敲除猪的系谱(图9),现在已繁育到第3代。表明本发明通过编辑ASGR1基因序列可制备稳定繁育的基因编辑动物种群。
序列表
<110> 成都中科奥格生物科技有限公司
<120> ASGR1突变基因在制备拟人化的低血脂代谢动物模型中的应用
<130> MP2000056
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> 人工序列(Artificial sequence )
<400> 1
agcagtttgt gtccgacctg cgg 23
Claims (10)
1.ASGR1突变基因在制备拟人化的低血脂代谢动物模型中的应用;其中所述ASGR1突变基因为ASGR1基因第5外显子上的两个等位基因第2817~2961位发生突变。
2.根据权利要求1所述应用,所述的ASGR1突变基因,其为
(1)ASGR1基因第5外显子上的两个等位基因第2840~2859位均发生20bp删除;或
(2)ASGR1基因第5外显子上的一个等位基因第2817~2961位发生146bp删除,另一个等位基因第2852~2853位增加1bp。
3.根据权利要求1所述应用,所述动物为猪。
4.根据权利要求1所述应用,所述拟人化的低血脂代谢为与人ASGR1基因突变产生的低血脂表型一致。
5.拟人化的低血脂代谢动物模型的制备方法,采用基因编辑技术制备具有ASGR1突变基因的动物模型。
6.根据权利要求5所述的制备方法,所述的ASGR1突变基因,其为
(1)ASGR1基因第5外显子上的两个等位基因第2840~2859位均发生20bp删除;或
(2)ASGR1基因第5外显子上的一个等位基因第2817~2961位发生146bp删除,另一个等位基因第2852~2853位增加1bp。
7.根据权利要求5所述的制备方法,所述基因编辑技术为CRISPR/Cas9基因编辑技术。
8.根据权利要求7所述的制备方法,利用CRISPR/Cas9基因编辑技术制备具有ASGR1突变基因的动物模型的方法具体为在ASGR1基因第5外显子设计并合成特异识别靶序列DNA的sgRNA,构建含有sgRNA的表达载体用于细胞转染受体细胞,核移植制备动物胚胎,移植到发情的受体动物子宫内制备。
9.根据权利要求6所述制备方法,所述sgRNA的序列如SEQ ID NO:1所示。
10.根据权利要求7所述制备方法,所述表达载体为PX330表达载体。
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