CN111019970A - Ndufa13在制备自发性肝炎-肝纤维化动物模型、及制备药物中的应用 - Google Patents
Ndufa13在制备自发性肝炎-肝纤维化动物模型、及制备药物中的应用 Download PDFInfo
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Abstract
本发明提供NDUFA13及其基因作为肝炎或肝纤维化药物或诊断靶点的用途。本发明的研究结果显示,NDUFA13及其基因与肝炎或肝纤维化密切相关,其为后期肝炎或肝纤维化药物和诊断试剂的研发提供了一个全新的靶点以及新的诊断、预防、治疗手段和思路。同时,本发明首次构建NDUFA13 flox/‑Alb‑Cre小鼠品系,该肝特异性NDUFA13杂合敲除小鼠可稳定出现非感染性自发性肝脏炎症,并在后期进展为肝纤维化表型,可作为自发性肝炎‑肝纤维化小鼠模型,应用于肝脏炎症‑肝纤维化机制研究及新药研发评价等。
Description
技术领域
本发明涉及生物技术领域,特别涉及NDUFA13在制备药物、诊断试剂及构建动物模型的应用。
背景技术
慢性肝病是一类临床慢性肝脏疾病谱的总称,包括各种诱导形成的肝炎、肝硬化以及肝癌等,其发病率与死亡率位居世界前列,我国约3亿人受到肝病影响,每年肝硬化引起全球约120万人死亡,严重危害人类健康。许多因素与慢性肝病发生发展有关如病毒感染、自身免疫反应、过敏反应、药物治疗、辐射等,这些因素可诱导慢性肝病的“肝炎-肝硬化-肝癌”病理三部曲进程,其中肝纤维化(Liver fibrosis,LF)作为慢性肝病进展的关键阶段,是众多慢性肝病进展为肝硬化的共同的病理学改变,若不及时有效干预,将导致不可逆的肝硬化甚至肝癌。由于目前尚无安全有效的临床干预手段,亦缺乏直接预防或逆转纤维化的治疗药物,使得肝纤维化成为长期以来慢性肝病临床治疗的难点。
由于医学伦理限制,使得疾病动物模型成为模拟人类疾病发生发展研究的重要手段,也是各种干预措施实施的重要实验对象。现阶段,模拟人肝纤维化病理进程的疾病动物模型目前主要集中于非遗传性与遗传性诱导两个方面。一是化学诱导、饮食诱导、感染因素诱导以及外科诱导等非遗传性肝纤维化模型。如四氯化碳、二甲基亚硝胺以及酒精等化学诱导、高脂饮食诱导、外科处理造成的胆管阻塞性诱导肝纤维化模型。而这些诱导模型具有较大局限性。如这些诱导物作用与肝细胞的具体分子靶点大多不清楚;各类模型在造模周期、途径及生物安全等方面均具有一定的局限性,如化学性模型死亡率较高,纤维化程度不易控制且药物具有致癌性等,酒精性与免疫性模型周期较长,胆管阻塞性模型存在手术复杂、模型长期存活率较低等系列问题。二是基因敲除缺陷遗传性诱导模型。如多药耐药相关蛋白2(Mdr2)基因敲除缺陷小鼠,可表现为原发性硬化性胆管炎与胆汁型肝纤维化表型,并在短期的4-6周龄时发生肉眼可见的肿瘤结节。又如ALMS1 Fat ausi基因突变小鼠,经高脂长时间饲养诱导,可发展为不可逆性肝纤维化表型。由于上述两种基因工程鼠均为全身性基因突变,尤其是上述两种基因在人类肝纤维化病理过程中是否存在缺陷尚不明了,且严重程度上以及病理进展与人肝纤维化具有较大差异,故在临床转化应用中存在缺陷。因此,开发一种与人肝纤维化疾病临床紧密相关的肝纤维化小鼠模型,不仅可极大丰富肝纤维化模型,为肝脏疾病的治疗与干预提供一种理想的实验动物模型。
线粒体呼吸链复合体I结构蛋白NDUFA13(NADH dehydrogenase(ubiquinone)Ⅰalpha subcomplex 13),又称为GRIM-19蛋白,是线粒体呼吸链(MRC)复合体Ⅰ的必须组份,其基因主要定位于人19号染色体19p13.2,编码144个氨基酸的蛋白质分子,分子量约为16kDa,主要定位于线粒体内膜,亦有少部分存在于细胞质与细胞核中,在线粒体MRC复合体膜电位、线粒体呼吸氧化磷酸化功能维持、细胞凋亡及多种信号转导调控中具有重要作用。近年研究发现NDUFA13作为一种抑癌基因在结直肠癌、前列腺癌、肝癌、胃癌、胶质瘤等多种肿瘤表达缺失,与肿瘤发生发展相关。也有研究报道NDUFA13缺失与“炎症-萎缩-肠上皮化生”的人类慢性萎缩性胃炎病理进程密切相关。尽管NDUFA13在肝癌中显著缺失,但NDUFA13与肝癌前期病理进程如肝炎、肝纤维化、肝硬化等的关系并不清楚。
发明内容
本发明首次公开了NDUFA13基因缺陷与肝炎及肝纤维化的病理进程相关,其肝脏的缺失可直接诱导自发性的非感染性肝炎,并可进一步进展为肝纤维化。
本发明的目的在于提供NDUFA13及其基因作为肝炎或肝纤维化药物或诊断靶点的用途。本发明的研究结果显示,NDUFA13及其基因与肝炎或肝纤维化密切相关,其为后期肝炎或肝纤维化药物和诊断试剂的研发提供了一个全新的靶点以及新的诊断、预防、治疗手段和思路。
为了实现上述发明目的,本发明提供以下技术方案:
NDUFA13作为药物靶点或诊断靶点在制备治疗、预防、诊断肝炎或肝纤维化药物或试剂中的应用。
上述药物或试剂是指调控或检测NDUFA13的表达水平的药物或试剂。
优选的,上述药物是增强NDUFA13的活性的药物。
NDUFA13基因作为药物靶点或诊断靶点在制备治疗、预防、诊断肝炎或肝纤维化药物或试剂中的应用。
靶向NDUFA13的增强剂在制备治疗或预防肝炎或肝纤维化药物的药物中的应用。
本发明首次构建NDUFA13flox/-Alb-Cre小鼠品系,该肝特异性NDUFA13杂合敲除小鼠可稳定出现非感染性自发性肝脏炎症,并在后期进展为肝纤维化表型,可作为自发性肝炎-肝纤维化小鼠模型,应用于肝脏炎症-肝纤维化机制研究及新药研发评价等。
本发明的目的还提供了NDUFA13及其基因在构建肝炎/肝纤维化动物模型中的应用。
为了实现上述发明目的,本发明提供以下技术方案:
NDUFA13基因在非感染性肝炎(自发性肝炎)或肝纤维化动物模型中的应用。NDUFA13基因的肝脏特异性缺失成功构建了肝炎肝纤维化动物模型。
本发明还提供了所述非感染性肝炎或肝纤维化动物模型的构建方法。
一种动物模型的构建方法,其特征在于,应用NDUFA13flox/-小鼠及Alb-cre小鼠品系,杂交构建NDUFA13flox/-Alb-cre小鼠品系。
本发明利用Cre/loxP转基因技术进行了肝纤维化疾病模型的建立,首次将NDUFA13 flox基因编辑小鼠NDUFA13flox/flox与Alb-Cre基因编辑小鼠杂交,得到肝脏特异性NDUFA13杂合敲除小鼠品系(NDUFA13flox/-Alb-Cre,SPF级)。
一种动物模型的构建方法,包括以下步骤:
(1)确定目的基因:小鼠NDUFA13(GenBank accession number:NM_023312.2:,Ensembl:ENSMUSG00000036199)位于小鼠第8号染色体,确定了5个外显子(自外显子2中ATG起始密码子至外显子5中TAG终止密码子),选择外显子3作为条件性敲除区域。外显子3的删除将导致NDUFA13基因功能的缺失。
(2)设计与构建打靶载体质粒:使用来自C57BL/6J文库的BAC克隆RP23-74A9或RP23-114L20作为模板,通过PCR产生同源臂和CKO区域。在打靶载体中,Neo盒的侧翼为Frt位点,CKO区域的侧翼为LoxP位点。DTA将用于阴性选择。
(3)ES细胞电转和阳性克隆筛选后,进行ES显微注射和制备嵌合体,嵌合体小鼠与flp小鼠杂交,敲除NEO抗性基因,获得NDUFA13flox/-F1小鼠。繁殖获得后代小鼠并鉴定,与野生型小鼠C57B/L6合笼繁殖传代。
(4)将NDUFA13flox/-小鼠与Alb-cre小鼠合笼,鉴定获得NDUFA13flox/-Alb-cre小鼠,建立NDUFA13flox/-Alb-cre小鼠品系。
上述方法用于肝特异性NDUFA13敲除小鼠诱导自发性肝炎与肝纤维化动物模型的构建。
有益效果
1.肝纤维化的致病因素众多,其进展的共同特征是炎症环境下损伤修复过程的异常调节。目前几乎没有直接预防或逆转纤维化的治疗药物。我们首次发现NDUFA13缺陷可诱导自发性、非感染性肝炎,并可进展为肝纤维化表型。
治疗和筛药不能直接用人来实验,需要多种具有针对性的动物病理模型。我们构建了肝脏特异性NDUFA13杂合敲除小鼠模型(NDUFA13flox/-Alb-cre肝脏特异性NDUFA13敲除小鼠),可诱发非感染性肝炎,并可进一步发展为肝纤维化,其病理进程与表型契合肝纤维化发展与机制,致病因素符合肝纤维化进展的共同特征,可应用于自发性肝炎-肝纤维化相关疾病诊断与治疗研究,是一种可广泛应用的肝炎肝纤维化动物模型。可用于肝脏炎症-肝纤维化机制研究、肝纤维化诊断标志物开发以及新药研发评价等。
2.本发明探究了NDUFA13在肝炎肝纤维化病理进程中的作用,NDUFA13基因参与肝炎及肝纤维化的发展,并成功构建了基于Cre/loxP系统小鼠肝纤维化模型,这对于肝炎肝纤维化的研究具有重要的现实意义。Cre/loxP系统源于F1噬菌体,通过Cre重组酶特异性识别loxP位点并切割位于2个loxP之间的DNA序列,达到敲除某个基因的目的。该方法可实现对某种组织特定基因的敲除,对小鼠基因组的修饰处在时空可调控状态,具有敲除效率高、应用范围广、胚胎致死率低等优点。目前已创建有许多器官特异性Cre和/或诱导型Cre转基因小鼠系,白蛋白(ALB)启动子是只表达在肝中的Cre重组酶,但是基于Cre/loxP系统开发的肝病相关动物模型极少。
3.与同龄同窝对照组相比,本发明所构建的模型小鼠肝脏中CK-18阳性的肝细胞中部分NDUFA13蛋白缺失,在4周龄时肝组织即出现肝窦扩张、肝小叶结构紊乱等肝慢性损伤病理表现。与同龄对照组相比,4周龄与近24月龄的NUDFA13杂合敲除小鼠均伴随CD45+、F4/80+以及MPO+炎症细胞浸润且24月龄小鼠炎症更为明显,24月龄NUDFA13杂合敲除小鼠肝脏IL-33、IL-1β、TNF-α等炎症因子表达均明显增多。这些表型证据体现出炎症具有自发性、持续性、进展性的反应特点。
后期的肝纤维化表现为一种进展性的表型特点。4周龄时肝纤维化特异性Masson三色染色基本为阴性,而近24月龄的NUDFA13杂合敲除小鼠肝组织内Masson染色呈强阳性,且纤维化促进相关因子TGF-β、Collagen-I、Collagen-III与TIMP1表达明显上调,而纤维化抑制相关因子MMP-9表达明显降低,表明肝脏明显胶原沉积,出现明显纤维化病理改变。与同龄对照组及4周龄NUDFA13杂合敲除相比,24月龄NUDFA13杂合敲除小鼠小鼠肝星形细胞(HSCs)活化标志物α-SMA表达明显上调,表明肝星形细胞(HSCs)大量激活。这些证据体现出早期主要为慢性炎症但非肝纤维化特点,而在后期表现为持续的炎症与肝纤维化表型特点。
4.本发明所构建的肝炎/肝纤维化动物模型呈现以下优势:
(1)疾病表型出现早(4w)、持续时间长(至少2年)、并且稳定性好(短时间内未发现向肿瘤等转化),与人类肝纤维疾病进展慢性病程进展相似。
(2)易于饲养繁殖,按照常规方法即可饲养,无需特殊饲料或化合物处理,与非条件性转基因小鼠及其他肝纤维化动物模型相比,操作安全、简便。
(3)该小鼠为基因稳定敲除导致的基因缺失,若该基因缺失未得到有效纠正,由该基因缺失所导致的表型不会发生自行逆转。
(4)特异性好,仅在肝脏特异性敲除。制备效率高,仅肝脏发生相应病变,早期阶段其它器官以及全身未发现异常,能较好反映人类肝纤维化的临床特点。
(5)具有慢性炎症-肝纤维化发展的慢性病理进程特点。所有NUDFA13杂合敲除小鼠肝脏在早期4周时均出现轻度炎症表型,在1.5-2年慢性进展为肝纤维化表型并伴有严重的炎症表型,存活时间至少2-3年,能较好反映人类肝纤维化慢性病程的临床特点。
(6)NDUFA13flox/-小鼠与Alb-cre小鼠可稳定繁育,合笼所获得的NDUFA13flox/-Alb-cre小鼠品系亦可大量繁殖且表型稳定,无致死性及其他疾病。
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示重组靶向载体构建图谱;
图2示靶向载体电转后基因重组;
图3示重组体鉴定引物设计策略;
图4示重组克隆PCR鉴定结果;其中A为3’Arm PCR鉴定结果,B为LoxP Site PCR鉴定结果;
图5示胚胎注射后,嵌合体小鼠与flp小鼠杂交,敲除NEO抗性基因,获得F1子代;
图6示F1子代LoxP Site PCR鉴定结果;
图7示NDUFA13flox/-Alb-cre小鼠基因型鉴定结果;
图8示NDUFA13flox/-小鼠与Alb-cre小鼠合笼后所获得的NDUFA13flox/-Alb-cre小鼠基因编辑结果;
图9示NDUFA13flox/-Alb-cre小鼠大体及解剖结果;
图10示小鼠肝脏NDUFA13与CK-18双标免疫荧光结果;
图11示CD45、F4/80、MPO免疫组化分析结果;
图12示肝脏炎症因子表达分析结果;
图13示小鼠肝脏纤维化特异性Masson染色结果;
图14示肝脏纤维化相关因子TGF-β、CollagenⅠ、CollagenⅢ、TIMP-1、MMP-9表达分析结果;
图15示肝脏肝星形细胞活化情况分析结果。
具体实施方式
本发明公开了一种基因的应用及动物模型的构建方法,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。本发明的方法及应用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和应用进行改动或适当变更与组合,来实现和应用本发明技术。
本发明提供的基因的应用及动物模型的构建方法中所用原料及试剂均可由市场购得。
下面结合实施例,进一步阐述本发明:
实施例1设计与构建NDUFA13打靶载体质粒
1、用高保真度DNA聚合酶从BAC克隆扩增小鼠基因组片段,并将其与重组位点和选择标记一起组装成靶向载体,如图1所示。
实施例2转基因
1、将靶向载体电转至C57BL/6ES细胞中,选取96个(一个96孔板)G418抗性克隆。基因重组结果如图2所示。PCR筛选策略如图3所示。采用mNdufa_3'PCR_F/mNdufa_3'PCR_R和mNdufa_LoxP_F/mNdufa_LoxP_R引物进行PCR测定。具体如下:
(1)3’Arm PCR引物序列
mNdufa_3’PCR_F:GCTGACCGCTTCCTCGTGCTTTA |
mNdufa_3’PCR_R:ACCATGCCTGGAATGAGACCCTAT |
得到的片段大小为3.7kb。 |
(2)LoxP Site PCR引物序列
如图4A所示,经3’Arm PCR引物鉴定共有21个阳性克隆:1A2,1C2,1F1,1F2,1G1,1G2,1H2,1A3,1B3,1C3,1D3,1E4,1G3,1G4,1H3,1C5,1C6,1F5,1G5,1H5和1H6。将上述21个阳性克隆经LoxP Site PCR引物鉴定,如图4B所示,共有9个阳性克隆:1A2,1F2,1A3,1D3,1E4,1G4,1F5,1H5和1H6。
2、ES显微注射,制备代孕鼠后胚胎移植,获得嵌合体小鼠。如图5所示,将1D3嵌合体小鼠与flp小鼠杂交,敲除NEO抗性基因,共产生10只F1子代,如图6所示,LoxP Site PCR鉴定第1,2,4,6,8号为Loxp-NDUFA13阳性,3’Arm PCR再次确认,最后,3只雄性(1,2,6号)和2只雌性(4和8号)F1杂合突变小鼠被确认为Loxp-NDUFA13携带体小鼠,与C57B/L6合笼繁殖,进行下一步实验。
实施例3 NDUFA13flox/-Alb-cre小鼠品系合笼繁育与鉴定
1、NDUFA13flox/-Alb-cre小鼠繁育
(1)将上述NDUFA13flox/-小鼠与Alb-cre小鼠合笼,获得NDUFA13flox/-Alb-cre小鼠。
2、NDUFA13flox/-Alb-cre小鼠基因型鉴定
(1)穿戴好无菌手套、口罩、帽子、无菌服后进入SPF级动物实验室。使用灭菌器械为小鼠打上耳标,并取3-5mm鼠尾至对应编号的1.5ml Ep管内,加入200uL lysis buffer(10μm Tris-HCl pH8.0,10μm EDTA、15mM NaCl,0.5%SDS)及4uL蛋白酶k,55℃恒温消化过夜。14000g离心10min取100uL上清与等体积异丙醇混匀,涡旋振荡10-20s,14000g离心15min,弃上清后加入70%酒精清洗沉淀,涡旋振荡10-20s,14000g离心10min后弃上清,室温30min左右晾干,加入100uL ddH2O或TE水,溶解获得DNA溶液。
配制相应PCR反应体系,并选择相应的程序进行反应。之后,琼脂糖凝胶电泳进行条带分析。
(2)PCR引物序列
LoxP Site PCR引物序列:
Alb-cre PCR引物序列:
(3)PCR引物反应体系
LoxP Site PCR反应体系(20uL):
Alb-cre PCR反应体系(12uL):
反应组分 | 体积(uL) | 终浓度 |
ddH<sub>2</sub>O | 4.25 | |
5X Kapa 2G HS buffer | 2.40 | 1X |
25mM MgCl<sub>2</sub> | 0.96 | 2mM |
10mM dNTP KAPA | 0.24 | 0.2mM |
20uM P1 | 0.30 | 0.5uM |
20uM P2 | 0.30 | 0.5uM |
20uM P3 | 0.30 | 0.5uM |
5mM 10x Loading Dye | 1.20 | 0.5mM |
2.5U/uL Kapa 2G HS taq polymerase | 0.05 | 0.01U/uL |
模板DNA | 2.00 |
(4)PCR扩增条件
LoxP Site PCR扩增条件:
Step# | Temp℃ | Time | Note |
1 | 94 | 2min | |
2 | 94 | 30sec | |
3 | 60 | 30sec | |
4 | 72 | 1min | |
5 | 30Times to 2 | ||
6 | 72 | 5min | |
7 | 4 | 99hrs | |
8 | End |
Alb-cre PCR扩增条件:
利用琼脂糖凝胶电泳鉴定PCR产物,结果图7为NDUFA13flox/-Alb-cre小鼠基因型的鉴定。如图8所示,该小鼠肝脏表达cre重组酶,识别LoxP位点,NDUFA13基因3号外显子被敲除。如图9所示,该NDUFA13基因杂合敲除小鼠至24月龄时与同龄WT小鼠比较外观无缺陷,除肝脏外其他器官亦无异常。
实施例4 NDUFA13蛋白表达分析
利用CK-18与NDUFA13双标免疫荧光技术,分析检测CK-18阳性的肝细胞中NDUFA13蛋白敲除情况,如图10所示,该小鼠肝脏中CK-18阳性的肝细胞中部分NDUFA13蛋白缺失,这与NDUFA13基因杂合敲除的基因分型相符。
实施例5肝脏内炎症细胞浸润分析
利用免疫组化技术,分析检测CD45、F4/80、MPO等炎症免疫细胞标志物在4周龄与24月龄NDUFA13基因杂合敲除小鼠肝脏表达情况。如图11所示,该4周龄小鼠肝脏CD45、F4/80、MPO表达均明显增多;尤其在24月龄小鼠肝脏表达更为明显;表明明显的炎症细胞浸润。
实施例6肝脏炎症因子表达分析
利用免疫组化技术,分析检测IL-33、IL-1β、TNF-α等炎症因子在24月龄NDUFA13基因杂合敲除小鼠肝脏表达情况。如图12所示,与同窝同龄对照小鼠肝脏相比,NDUFA13基因杂合敲除小鼠IL-33、IL-1β、TNF-α等炎症因子表达均明显增多,表明显著的炎症因子释放。
实施例7肝纤维化表型检测与纤维化相关因子分析
利用纤维化特异性Masson三色染色方法检测小鼠肝脏纤维化情况。如图13所示,4周龄Masson三色染色基本为阴性,24月龄NDUFA13基因杂合敲除小鼠肝脏Masson三色染色显著阳性。利用免疫组化染色方法检测纤维化相关因子TGF-β、Collagen-I、Collagen-III、TIMP1、MMP-9的表达。如图14所示,在24月龄NUDFA13杂合敲除小鼠纤维化的肝组织内,纤维化促进相关因子TGF-β、Collagen-I、Collagen-III与TIMP1表达明显增强,而纤维化抑制相关因子MMP-9表达明显降低。
实施例8肝纤维化相关的肝星形细胞(HSCs)活化分析
利用免疫组化染色方法检测肝星形细胞(HSCs)活化标志物α平滑肌肌动蛋白(α-SMA)表达情况,并利用免疫荧光双标技术检测Desmin(HSCs标志物)与α-SMA共表达情况。如图15所示,在24月龄NUDFA13杂合敲除小鼠,肝组织内HSCs活化标志物α-SMA表达明显上调,但在4周龄小鼠肝脏基本为阴性;而HSCs中Desmin与α-SMA明显共表达,表明肝星形细胞(HSCs)大量激活。
综上所述:本发明构建的肝特异性NDUFA13敲除小鼠诱导自发性肝炎与肝纤维化动物模型构建成功。
以上所述仅是本发明的优选实施方式,其中实施例1与例2由赛业生物科技有限公司代为完成。应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (9)
1.NDUFA13作为药物靶点或诊断靶点在制备治疗、预防、诊断肝炎或肝纤维化药物或试剂中的应用。
2.根据权利要求1所述的应用,所述药物或试剂是指调控或检测NDUFA13的表达水平的药物或试剂。
3.根据权利要求1所述的应用,所述药物是增强NDUFA13活性的药物。
4. NDUFA13基因作为药物靶点或诊断靶点在制备治疗、预防、诊断肝炎或肝纤维化药物或试剂中的应用。
5.靶向NDUFA13的增强剂在制备治疗或预防肝炎或肝纤维化药物的药物中的应用。
6.NDUFA13基因在构建非感染性肝炎或肝纤维化动物模型中的应用。
7.一种动物模型的构建方法,其特征在于,应用NDUFA13flox/-小鼠及Alb-cre小鼠品系,杂交构建NDUFA13 flox/-Alb-cre小鼠品系。
8.如权利要求7所述的动物模型的构建方法,包括以下步骤:
(1)确定目的基因:小鼠NDUFA13基因的外显子3作为条件性敲除区域;
(2)设计与构建打靶载体质粒,使用来自C57BL/6J文库的BAC克隆RP23-74A9或RP23-114L20作为模板,通过PCR产生同源臂和CKO(conditional knock out)区域;在打靶载体中,Neo盒的侧翼为Frt位点,CKO区域的侧翼为LoxP位点;
(3)ES细胞电转和阳性克隆筛选,进行ES细胞显微注射和制备嵌合体,嵌合体小鼠与Flp(Flippase)小鼠杂交,敲除NEO抗性基因,获得NDUFA13 flox/ - F1小鼠;繁殖获得后代小鼠并鉴定,与野生型小鼠C57B/L6合笼繁殖传代;
(4)将NDUFA13 flox/ -小鼠与Alb-cre小鼠合笼,鉴定获得NDUFA13 flox/ -Alb-cre小鼠,建立NDUFA13 flox/ -Alb-cre小鼠品系。
9.如权利要求7或8所述动物模型的构建方法用于自发性肝炎与肝纤维化动物模型的构建。
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