CN111705063B - Asgr1突变基因及其在制备哺乳动物肝损伤敏感模型中的应用 - Google Patents
Asgr1突变基因及其在制备哺乳动物肝损伤敏感模型中的应用 Download PDFInfo
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Abstract
本申请属于基因工程技术领域,公开了一种基因工程编辑位点以及获得的ASGR1突变基因在制备哺乳动物肝损伤敏感模型中的应用。通过对ASGR1基因序列中合适的编辑位点进行基因编辑,获得了传代稳定、易于诱导肝损伤的动物模型,并建立了稳定的繁育群体。本发明建立了一种获得传代稳定、易于诱导肝损伤的基因工程非人哺乳动物模型群体的方法,满足各种的实验需求。
Description
技术领域
本发明属于基因工程技术领域,具体涉及ASGR1突变基因及其在制备哺乳动物肝损伤敏感模型中的应用。
背景技术
动物疾病模型是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,而且许多实验在实验动物伦理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。
肝损伤是各种肝脏疾病的病变结果,对肝损伤的防治目前仍是一个严峻的课题。通过建立实验性肝损伤动物模型,研究肝病的发生机制,筛选保肝药物,探索保肝作用原理,具有重要的现实意义。肝损伤模型按成因可分为酒精肝损伤、药物性肝损伤、病毒性肝损伤。
现有的制备技术主要有三类:
1)外科手术制备。通过手术的方式,切除部分器官,造成器官损伤。由于动物个体间差异难以做到表型完全一致、手术操作及护理等问题限制了其应用。
2)药物诱导。通过激素、化学试剂的方法造成动物,内循环受阻、脏器损伤。药物诱导产生模型较外科法有针对性,且种类范围更广,但是由于药物价格及剂量原因,在大动物上应用较少,且同样由于动物个体间差异,难以做到表型完全一致,无法满足规模制备和应用。
3)基因编辑技术制备动物疾病模型。通过基因工程技术来制备疾病模型,与外科手术制备和药物诱导2种方法相比,基因编辑技术可以做到表型一致。但是基因编辑技术的关键在于需要筛选到合适的候选基因及编辑位点,且基因编辑动物尤其是疾病模型类的基因编辑动物的存活率低等限制了其发展。
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,用于高效制备易于诱导、可自然传代的非人哺乳动物肝损伤模型。
为实现本发明的目的,本发明采用如下技术方案:
一种ASGR1突变基因其为ASGR1基因第5外显子上的两个等位基因第2840~2859位发生突变。
突变是指生物体、病毒或染色体外DNA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。
去唾液酸糖蛋白受体1(ASGR1)广泛表达在肝实质细胞表面,主要功能是清除血液循环系统中的糖蛋白、凋亡细胞以及脂蛋白等,其相关研究主要集中于靶向肝脏药物递送的应用及对于异种肝脏(细胞)移植术后产生的血小板减少性内出血。申请人模拟部分ASGR1基因突变人群对大动物进行基因ASGR1的编辑,获得易于诱导成不同类型肝损伤模型、且可以正常繁育的大动物群体。因此本发明还提供了所述ASGR1突变基因在制备哺乳动物肝损伤敏感模型中的应用。
其中ASGR1突变基因其为ASGR1基因第5外显子上的两个等位基因第2817~2961位发生突变。
在一些实施方案中,所述ASGR1突变基因为(-20bp/-20bp)突变型或(-146bp/+1bp)突变型。
在本发明中,所述哺乳动物为猪。在一些实施方案中,所述哺乳动物为巴马小型猪。
在本发明中,所述肝损伤为酒精性肝损伤或药物性肝损伤。在一些实施方案中,所述药物性肝损伤为四氯化碳诱导肝损伤。
进一步的,本发明还提供了哺乳动物肝损伤敏感模型的制备方法,采用基因编辑技术制备具有所述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CRISPR/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,包括锌指核酸内切酶(Zinc figernucleases,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 effectornuclease,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表达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,本发明提供了一种基因工程编辑位点以及获得的ASGR1突变基因在制备哺乳动物肝损伤敏感模型中的应用。通过对ASGR1基因序列的合适的编辑位点进行基因的编辑编辑获得的动物易于诱导成不同的肝损伤模型、且可以正常繁育,建立了稳定的繁育群体,满足规模化的使用需求,用于不同的实验。本发明还建立了一个获得易于诱导成不同肝损伤、可自然传代的基因工程非人哺乳动物模型群体的制备方法,可用于肝损伤模型的规模化制备。
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1ASGR1基因sgRNA靶点设计示意图;
图2示实施例1ASGR1基因敲除载体设计示意图;
图3示实施例1ASGR1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2ASGR1基因敲除仔猪肝脏组织病理学检测结果图;
图5示实施例3ASGR1基因敲除仔猪系谱图;其中F0:原代猪;F1:F1代杂合子猪。
具体实施方式
本发明公开了ASGR1突变基因及其在制备哺乳动物肝损伤敏感模型中的应用。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基因的失活,会影响肝脏功能,失活该基因的动物经过酒精及四氯化碳诱导,会较WT动物更容易产生肝脏纤维化等损伤表型。本发明我们发现并建立了一个可高效建立不同肝损伤模型的基因工程非人哺乳动物的制备方法,可用于不同肝损伤模型的规模化制备。
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6外显子(图1)设计并合成了特异识别靶序列DNA的sgRNA(sgRNA:agcagtttgtgtccgacctgcgg,SEQ ID NO:1)。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 GreenFluorescentProtein,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3种不同突变类型的(-20bp/-20bp,-146bp/+1bp)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263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western blot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)。Westernblot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基因敲出仔猪对肝损伤的敏感性
对获得的ASGR1基因敲除猪进行了2种肝损伤方式诱导。
1、试验动物
实验组:ASGR1基因敲除猪(ASGR1敲除猪与WT母猪配种繁育产生的个体)后代。
对照组:以未进行基因编辑的野生型猪(wide type,WT)作为对照。
2组动物均为6月龄、性别、饲喂与环境均相同。
2、试验方法:
肝损伤诱导方式:(1)以50%浓度酒精作为饮水替代物,饲喂猪2个月,模拟人饮酒产生酒精性肝损伤;(2)腹腔注射四氯化碳15天,模拟人药物性肝损伤。
血液检测肝损伤:诱导前采血取血清,然后检测血液生化指标,检测指标:ALT,AST,GTT,TBIL,ALB,A/G,TP;实验开始后,在注射四氯化碳3天后抽血1次,检测同样的指标;实验开始1周后,抽血检测同样指标;直到肝功能指标不再升高时采集肝脏样品。(或者AST升高到正常水平的三倍以上)
病理切片分析:对诱导后的ASGR1敲除猪及对照(WT)进行了肝脏病理学分析,采用HE染色分析肝脏是否出现细胞凋亡产生的空洞及免疫细胞聚集、采用Massson染色检测肝部有无纤维化病变。
3、试验结果:
对ASGR1敲除猪及对照(WT)进行酒精饲喂及四氯化碳注射后检测其血液生理化学指标,结果见表2
表2对猪血液生理化学指标的影响
结果显示,与WT个体相比ASGR1敲除猪与肝功能相关的指标谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)、谷氨酰转移酶(GGT)、尿素(UREA)出现异常升高,且差异显著。
对诱导后的ASGR1敲除猪及野生型猪进行了肝脏病理学分析,采用HE染色分析肝脏是否出现细胞凋亡产生的空洞及免疫细胞聚集、采用Massson染色检测肝部有无纤维化病变。结果见图4
结果显示,酒精诱导的WT个体肝部未见明显异常,但ASGR1敲除猪的肝脏组织出现了部分空泡化,但程度较轻,二者均未见有免疫细胞聚集。Massson染色发现二者肝部均出现纤维化症状(蓝色),但ASGR1敲除猪纤维化更为显著。
四氯化碳诱导的个体肝脏组织均出现空泡化、且ASGR1敲除猪肝脏明显出现了免疫细胞聚集,揭示有炎症伴生。Massson染色发现二者肝脏组织均出现纤维化症状(蓝色),但ASGR1敲除猪效果纤维化更为显著。
综上所述,通过血液生化的检测及病理切片的分析,ASGR1敲除猪比野生型个体更易诱导产生肝损伤表型、且诱导方式不同、产生的损伤程度不同。
实施例3、ASGR1基因敲除仔猪可正常传代。
以ASGR1敲除猪为例,通过基因编辑的方法,获得了6头原代个体。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5),现在已繁育到第3代。表明本发明通过编辑ASGR1基因序列可制备正常繁育稳定遗传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突变基因在制备哺乳动物肝损伤敏感模型中的应用
<130> MP1936880
<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 (8)
1.一种ASGR1突变基因,其为ASGR1基因第5外显子上的两个等位基因第2817~2961位发生突变;
所述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。
2.权利要求1所述的ASGR1突变基因在制备哺乳动物肝损伤敏感模型中的应用;
所述哺乳动物为猪。
3.根据权利要求2所述应用,所述肝损伤为酒精性肝损伤或药物性肝损伤。
4.哺乳动物肝损伤敏感模型的制备方法,采用基因编辑技术制备具有权利要求1所述ASGR1突变基因的动物模型。
5.根据权利要求4所述的制备方法,所述基因编辑技术为CRISPR/Cas9基因编辑技术。
6.根据权利要求5所述的制备方法,利用CRISPR/Cas9基因编辑技术制备具有权利要求1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。
7.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。
8.根据权利要求6所述制备方法,所述表达载体为PX330表达载体。
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