CN111647656A - mir-29a基因在检测肝癌及肝纤维化中的应用及所述基因条件敲入小鼠的构建方法 - Google Patents
mir-29a基因在检测肝癌及肝纤维化中的应用及所述基因条件敲入小鼠的构建方法 Download PDFInfo
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Abstract
本发明提供mir‑29a基因在检测原发性肝细胞癌上的应用,以及mir‑29a基因在检测日本血吸虫肝纤维化的应用。本发明还提供mir‑29a基因条件敲入小鼠在研究原发性肝细胞癌和/或日本血吸虫肝纤维化上的应用。本发明证实mir‑29a在肝癌、癌旁以及肝癌细胞系中表达明显降低,这提示mir‑29a低水平表达可能与肝癌细胞的发生、发展相关。此外,miR‑29a条件性敲入小鼠可作为研究原发性肝细胞癌和慢性血吸虫感染或晚期血吸虫病引起的肝纤维化的动物模型,为后续进一步研究肝细胞癌和血吸虫病引起的肝纤维化药物提供依据和基础。
Description
【技术领域】
本发明一方面属于基因的功能与应用领域,特别涉及mir-29a基因在检测原发性肝细胞癌和日本血吸虫肝纤维化中应用;还涉及mir-29a基因条件敲入小鼠的构建方法及其应用。
【背景技术】
MiR-29家族(包括miR-29a、miR-29b、miR-29c)是由相应形状的前体 miRNA茎环结构或者pre-miRNA发夹结构剪切而来,多来自于pre-miRNA的3'(3p)端中的一条臂,少部分来自于其5'(5p)端。MiR-29家族分布在两条染色体上,miR-29b2和miR-29c分布在1q32,miR-29b1/miR-29a分布在7q32。三个家族成员中,miR-29a首先被Lagos-Quintana等人发现,紧接着大量的克隆研究揭示了成熟的miR-29a的产生。
肝细胞癌(Hepatocellular Carcinoma,HCC)为原发性肝癌中最常见的恶性肿瘤,约占其中的90%,在全球最常见的恶性肿瘤中排第六位,在癌症引起的死亡中排第二位。目前肝癌的治疗最有效的方法仍然是手术切除,然而,对于手术切除的肝癌患者,术后肿瘤复发与转移是影响患者长期生存的主要原因,目前仍没有有效的方法治愈。肝癌细胞的生长、侵袭和转移与肝癌的预后密切相关,因此只有深入研究肝癌生长、侵袭和转移的分子机制,才能找到治愈肝癌的治疗靶点。
miR-29的异常表达以及其作为癌基因或抑癌基因的功能已经在很多种肿瘤中广泛加以研究。通过对肿瘤组织或肿瘤细胞中表达的miRNA的转录分析研究发现,miR-29在大多数肿瘤中表达下调,多个研究已证明高表达miR-29可以引起肝癌细胞凋亡,而低表达miR-29增加HCC肿瘤形成和转移。一项新的研究发现,在大多数肿瘤中miR-29可下调癌基因或者上调抑癌基因,因此可诱导癌细胞凋亡、抑制癌细胞的增殖、侵袭。研究发现miR-29a与肿瘤关系密切,它在多种肿瘤中表达下调,但具体机制尚不清楚。
血吸虫病是由血吸虫引起的一种慢性寄生虫病,也是世界范围内威胁公众健康的主要问题之一,主要流行于中东、亚洲东部和南美洲等地。在我国,虽然血吸虫病疫情呈整体持续下降的态势,但是仍存在一定数量的血吸虫病传染源,其防控任务仍艰巨。据流行病学统计,我国现仍有6500万人口受到血吸虫的严重威胁,84万人患病,轻者丧失劳动能力,重者死亡。虫卵长期沉积和寄生虫免疫炎症损伤是发生特征性干线型肝硬化主要原因,肝纤维化过程中肝星状细胞 (Hepatic stellate cells,HSCs)活化是关键。HSCs被激活,并进行形态学和功能学的转分化,维生素A储存细胞被转换成收缩肌纤维母细胞,以应对肝脏损伤时细胞外基质(Extracellular matrix,ECM)的产生。随后,活化形式的HSC分泌促纤维化介质,例如转化生长因子(Transforming growth factor,TGF)-0并产生ECM 成分。纤维化的形态学特征是ECM蛋白分子的沉积增加,包括I/III型胶原,纤维连接蛋白和层粘连蛋白,所有这些都会加剧伤口愈合过程。I型纤维胶原由胶原-1α1(COL1α1)和胶原-1α2(COL1α2)编码,占健康肝脏ECM中总胶原的36%。在肝纤维发生的情况下,I型胶原是沉积在窦周区域的主要类型。然而,IV型胶原在正常肝脏中构成总胶原蛋白10%以下,肝纤维化时其表达量明显上调。调控活化的HSC中ECM基因表达的机制已成为肝纤维化潜在的治疗靶点。最近的研究表明,miR-29的水平在纤维化肝脏中显着降低,与人类肝硬化研究结果基本一致,其下调也会影响两种不同的纤维化动物模型(碳四氯化(CCL4)和胆管结扎(BDL))肝脏HSC的活化。TGF-0对HSC的刺激对肝纤维化的发生和进展是至关重要的,肝细胞、库普弗细胞和窦状内皮细胞分泌的TGF-0可导致HSC激活、转分化和分泌ECM。据报道,TGF-01能够介导HSC中miR-29 的下调;此外,miR-29在小鼠HSC过表达可导致胶原表达的下调,包括胶原 COL1 a1和COL4 a 1,此效应是通过直接靶向调控这些细胞EMC基因的mRNA 表达而发挥抗纤维化作用。现有研究已证实miR-29b-3p可以抑制日本血吸虫小鼠纤维化(Tao R.,Fan X.X.,YuH.J.et al.MicroRNA-29b-3p prevents Schistosoma japonicum-induced liverfibrosis by targeting COL1A1 and COL3A1[J].J Cell Biochem,2018,119(4):3199-3209.),但miR-29a在日本血吸虫肝纤维化的作用及相关机制尚有待理清。到目前为止,国际上无文献报道有关miR-29a在日本血吸虫肝纤维化中的功能或应用。
本发明旨在通过细胞、动物等体内外实验,多方面证实miR-29a在原发性肝细胞癌及日本血吸虫肝纤维化中作用,为临床诊疗提供新的思路和靶点;并且制备国内尚无成功建立和应用的mir-29a基因高表达小鼠,为以上疾病乃至更广泛的领域提供科学研究手段。
【发明内容】
本发明的目的是为了克服现有技术的缺陷与不足,确定mir-29a基因在原发性肝细胞癌及日本血吸虫肝纤维化中的作用,提供一种mir-29a作为药物靶标在筛选治疗原发性肝细胞癌和日本血吸虫肝纤维化的药物中的应用,进而提供一种 mir-29a的激动剂在制备治疗原发性肝细胞癌和日本血吸虫肝纤维化的药物中的应用;此外,设计并制备mir-29a基因高表达小鼠,作为一定领域范围的科学研究手段之一。
为了实现上述目的,本发明以人肝组织(包括肝细胞癌、癌旁和正常肝组织)、人肝细胞系(L02)为实验对象,通过实时定量多聚酶链式反应(RT-PCR)方法检测mir-29a基因水平,结果表明与正常肝组织、正常肝细胞系(LO2)相比, mir-29a在肝癌、癌旁以及肝癌细胞系(HepG2、Huh7、SMMC7721)中表达明显降低,这提示mir-29a低水平表达可能与肝癌细胞的发生、发展相关。
基于此,本发明提供mir-29a基因在检测原发性肝细胞癌上的应用。
另一方面,本发明以日本血吸虫小鼠模型为实验对象,通过RT-PCR方法对肝组织和分离提纯的肝细胞、HSCs及kupffer细胞进行mir-29a基因水平检测,结果表明与对照组小鼠及同时期原代肝细胞、kupffer细胞相比,肝纤维化明显期mir-29a在HSCs中的表达水平显著降低,这提示mir-29a很可能在 HSCs中发挥作用而参与了日本血吸虫小鼠的肝纤维化进展。
因此,本发明还提供mir-29a基因在检测日本血吸虫肝纤维化的应用。
此外,本发明通过体内外实验全面探讨mir-29a在原发性肝细胞癌和日本血吸虫肝纤维化HSCs中的作用,为临床诊断的靶向治疗提供有意义的线索和依据。
进一步地,本发明也利用CRISPR/Cas介导的基因工程技术制备mir-29a基因条件性敲入小鼠,并通过RT-PCR方法对此基因型进行验证和确认。本发明的 mir-29a基因敲入小鼠可与特异性白蛋白(Albumin)基因启动子驱动的Cre重组酶转基因小鼠(Alb-Cre+)杂交,从而得到某些脏器mir-29a特异性高表达的小鼠;拓展了有关mir-29a基因功能的研究范围,例如深入发掘mir-29a基因的调控分子,以寻找更加有效、可行的临床治疗靶点和治疗性分子。
因此,本发明还提供mir-29a基因条件敲入小鼠在研究原发性肝细胞癌和/或日本血吸虫肝纤维化上的应用。
本发明确认mir-29a基因的表达在检测原发性肝细胞癌和日本血吸虫肝纤维化中的,为后续研制该疾病的药物提供靶标。
【附图说明】
图1为实施例1中,人正常肝组织、肝细胞癌及癌旁组织标本中miR-29a 表达水平差异(*p<0.05,**p<0.01有意义);
图2为实施例1中,miR-29a在LO2、HepG2、Huh7、SMMC7721中的表达变化(*p<0.05有意义);
图3为实施例1中,Huh7细胞转染miR-29a mimics/inhibitor 50nm 48h 后miR-29a的表达以及其增值、侵袭和迀移能力的变化(*p<0.05,**p<0.01有意义)。
图4为实施例2中,各时间点日本血吸虫小鼠肝纤维化模型肝组病理染色;
图5为实施例2中,各时间点日本血吸虫小鼠肝纤维化模型肝组织和感染第8周肝脏3种原代细胞的miRNA29a-3p表达水平差异(*p<0.05,**p<0.01,***P<0.001有意义);
图6为实施例2中,体外转染miR-29a mimics对TGF-β1诱导活化的LX- 2细胞中纤维化指标的影响(##p或**p<0.01,###P或***P<0.001有意义);
图7为实施例3中,mir-29a基因条件性敲入小鼠的制备流程;
图8为实施例3中,靶载体验证;
图9为实施例3中,靶基因阳性表达小鼠检测(图9A&B)和测序(图9 C);
图10为实施例3中,阳性表达小鼠的Southern检测结果;
图11为实施例3中,H11-MIR29A-cKI小鼠的应用方式;
图12为实施例1中定量PCR程序。
【具体实施方式】
下面结合说明书附图和具体实施例对本发明进行详细阐述,所述实施例用于非限制性地解释本发明的技术方案。下述实施例中所使用的试验方法、材料及试剂等,如无特殊说明,均为常规方法或可从商业途径购买获得。
以下实施例涉主要及以下材料或设备:
1、实验用细胞及培养条件:
(1)人肝癌细胞系(HepG2、Huh7、SMMC7721)、人正常肝细胞系 (LO2)、人肝星状细胞系(LX-2)购买自中国典型培养物保藏中心(位于湖北省武汉大学)并由同济医院感染免疫疾病研究所保存;(2)小鼠原代肝星状细胞(HSCs)、Kupffer细胞、肝细胞为申请人在实验室根据现有技术的教导自行分离得到;(3)上述细胞均为贴壁细胞,其中,LO2、HepG2、Huh7、SMMC7721和LX-2在含有10%FBS的DMEM培养基中进行培养,HSCs在 20%FBS的DMEM培养基中进行培养,放置于37℃、体积浓度5%CO2培养箱中。
2、实验用动物及饲养条件:
(1)雄性Balb/c裸鼠、C57BL/6小鼠(6-8周、体重约20克左右)均购自湖南斯莱克景达实验动物有限公司;(2)Balb/c裸鼠饲养于同济医学院SPF级动物实验中心,C57BL/6小鼠饲养于P3级动物实验中心,H11-MIR29A-cKI小鼠饲养于赛业模式生物研究中心有限公司的SPF级动物实验中心;饲养条件:室温22-24℃、湿度45-70%、明暗交替灯照时间12小时、自由饮水摄食。
3、实验用钉螺:感染性野生钉螺受赠于自南京血吸虫病防治研究所。
4、主要抗体:兔源性Collagen I单克隆抗体、兔源性α-SMA抗体购自Abcam 公司,GADPH抗体购自Santa Cruz Biotechnology公司。
5、RT-PCR检测引物序列:
(1)miRNA RT-PCR反应与miRNA RT-qPCR反应所用特异性扩增引物均由广州锐博生物科技公司设计并合成;
(2)从NCBI(www.ncbi.nlm.nih.gov/GenBank)miRBase数据库中获取目的基因(Col 1α1、Col 3α1、a-SMA、TGF-β1、GAPDH)序列,选出同源性较高的序列作为引物模板;
表1:引物序列
实施例1 mir-29a在人肝癌组织、肝癌细胞系中的表达情况及对肝癌细胞的作用的分析
一、人正常肝组织/肝细胞癌/癌旁组织标本以及肝癌/正常肝细胞系中miR- 29a表达水平检测
收集人肝脏标本,共分为正常肝组织(n=12)、肝癌组(n=18)及癌旁组(n=18)。RT-PCR方法检测人正常肝组织、肝癌及癌旁组织标本中miR-29a表达水平,采用GraphPadPrism 5软件分析统计四组相对表达量之间有无显著性差异;上述条件下分别培养肝细胞系(L02、HepG2、Huh7、SMMC7721),应用RT-PCR方法检测正常肝细胞LO2,肝癌细胞系HepG2、Huh7、SMMC7721细胞中miR- 29a的表达水平。
其中,RT-PCR反应体系及程序设置如下:
(1)采用10μl体系
Forward Primer序列:F:5'-GGGTAGCACCATCTGAAAT-3'
Reverse Primer序列:R:5’-CAGTGCGTGTCGTGGAGT-3’
(2)在定量PCR仪上设定如图12所示程序:
结果如图1所示:18对肝细胞癌及癌旁组织比较,其中14对miR-29a在肝癌中的表达水平低于癌旁组织组,4对miR-29a在肝癌中的表达水平高于癌旁组织组 (如图1A所示);miR-29a在肝癌中的表达水平低于癌旁组织及正常肝组,癌旁组织中miR-29a水平低于正常肝组,且差异有统计学意义(如图1B所示,P<0.05),这可能和癌旁组织有肝纤维化或者肝硬化组织有关。
此外,图2显示,与正常肝细胞L02相比,肝癌细胞HepG2、Huh7、SMMC7721 中miR-29a的表达下调,且有统计学差异(P<0.05)。
综上所述,与癌旁组织、正常肝组织及正常肝细胞系比较,肝癌组织及肝癌细胞系中中miR-29a表达下调,说明miR-29a在肝癌中的异常表达可能与肝癌的发生发展、细胞增殖、血管新生及侵袭与转移过程密切相关。
二、miR-29a转染Huh7细胞并对其增值、侵袭和迀移能力的影响
选取huh7细胞进行干预,分为如下几组:miR-29a模拟物及miR-29a模拟物 NC组,miR-29a阻遏物及miR-29a阻遏物NC组,空白组。分别以miR-29a转染细胞48小时后,采用RT-PCR验证是否转入细胞内并检测miR-29a 1表达水平;同时应用CCK-8检测miR-29a对细胞增殖影响;应用transwell方法检测miR-29a转染后对肿瘤细胞侵袭和迀移的影响。
结果如图3所示,其中,转染miR-29a模拟物组与miR-29a模拟物NC组和空白组比较,结果显示miR-29a表达上调;转染miR-29a阻遏物组与miR-29a阻遏物NC 组和空白组比较,miR-29a表达下调,且均有统计学差异(如图3A,P<0.05)。CCK- 8检测发现在48小时和72小时时,过表达miR-29a抑制huh7细胞增殖;抑制miR- 29a则促进huh7细胞增殖,且差异具有统计学意义(如图3B,P<0.05)。另一方面,transwell显示,过表达miR-29a侵袭组和迀移组,huh7穿过小室的细胞数目减少,抑制表达miR-29a侵袭组和迀移组,huh7穿过小室的细胞数目增多,且均有统计学差异(如图3C和D,P<0.05)。
实施例2 mir-29a作用于肝星状细胞对日本血吸虫小鼠肝纤维化的影响
一、建立日本血吸虫小鼠肝纤维化模型
C57BL/6小鼠(6-8周),每组10只随机分成实验组(感染第4W、6W、8W、 12W)和对照组(感染第0W),感染操作步骤如下:(1)将含有尾蚴的钉螺置于光照下、水温水中,肉眼可见尾蚴逸出浮于水面。(2)以接种环前端挑取含尾蚴水少许置于盖玻片上,在解剖镜下计数(16±1条)。(3)除去小鼠腹部1×1cm2鼠毛,将玻片贴合于腹部15~20min。对照组以无菌生理盐水同样处理。(4)于P3级动物实验中心饲养。
二、模型鼠肝组织病理染色并检测mir-29a表达
分别在感染第0W、4W、6W、8W及12W时,麻醉处死后无菌操作台内留取小鼠肝组织和分离提纯肝细胞、HSCs及kupffer细胞,RT-qPCR检测各时间点的肝组织和3种细胞中miRNA29a水平,并且对固定包埋的肝组织切片做HE和Masson染色,观察胶原的生成情况。
图4显示,肝组织化学染色表明与对照组、感染第4W比,感染第8W和第12W 肝组织胶原生成以及肉芽肿面积明显增多;观察肝脏和脾脏外观,感染第8W和第 12W的肝脏体积明显缩小、颜色乌黑、表面凹凸不平、质地较硬,而脾脏明显增大。
图5显示,在日本血吸虫小鼠肝纤维化模型肝组织中,RT-qPCR结果为: miRNA29a表达先下调后上调,在感染第8W下调最明显,第12W有所上升,但仍明显低于对照组,且有统计学意义(如图6A,P<0.01)。感染第8W与对照组相比, miRNA29a在肝细胞和HSCs中下调显著(如6B,P<0.01),且在HSCs中下降最明显。
根据现有技术的教导,肝脏中血吸虫的聚集与其虫卵的沉积在肝脏微环境引起免疫和炎症反应可大量激活HSCs,导致细胞外基质(Extracellular matrix,ECMs)沉积,而免疫细胞的大量募集浸润进一步加重虫卵诱导的肉芽肿慢性炎症,最终围绕干支系统形成肝纤维化。本实施例的结果提示miRNA29a很有可能在HSCs中发挥调控作用从而参与了日本血吸虫小鼠的肝纤维化进展,为进一步探索日本血吸虫病肝纤维化发病机制提供理论依据。
三、外过表达和抑制miR-29a对LX-2细胞的影响
考察体外转染miR-29a mimics对TGF-β1诱导活化的LX-2细胞,实验分为:空白组、TGF-β1组、TGF-β1+NC模拟物组及TGF-β1+miR-29a模拟物组,转染48h后经RT-qPCR检测相关纤维化指标(Col 1α1、Col 3α1、α-SMA、TGF-β1)。
图6A显示荧光显微镜下观察结果,转染48h miRNA29a-3p模拟物可完全进入到LX-2细胞内;图5B显示体外转染RT-qPCR结果:与空白组比较,TGF-β1组和TGF-β1+NC模拟物组相关纤维化指标mRNA表达均明显上调(如图6B,P<0.01);与TGF-β1+NC模拟物组比较,TGF-β1+miR-29a模拟物组相关纤维化指标mRNA表达均显著下调(如图6B,P<0.01)。
本发明验证了mir-29a基因的表达水平与肝癌细胞的增殖、侵袭和迀移有关,也与日本血吸虫肝纤维化有关;因此,mir-29a可应用于肝癌细胞检测和血吸虫肝纤维化检测。
实施例3 mir-29a基因条件敲入小鼠(H11-MIR29A-cKI小鼠)制备及应用
一、mir-29a基因条件敲入小鼠(H11-MIR29A-cKI小鼠)制备
1、构建human MIR29A-H11-cKI donor载体
1)合成mir-29a基因序列(NCBI Reference Sequence:NR_029503.1),如SEQ No.1所示;
2)连接骨架的获取:构建H11位点基础骨架(Hipp11(H11)基因座位于小鼠 11号染色体上Eif4enif1和Drg1基因之间的基因间区域),并命名为VB027;使用AsiSI/PacI双酶切VB027载体骨架,胶回收11.5kb片段;
3)连接片段的获取:使用EcoRI酶切Mir-29基因合成产物合成质粒,胶回收片段;
4)片段+骨架使用In-fusion连接酶按照NEBuilder(E2621S)操作指南进行连接;
5)连接产物转化+涂板+过夜培养:在37℃条件下涂平板倒置培养16-18h;
6)挑选单菌落进行菌落PCR鉴定(F primer:TTAAGGGTTCCGGAT CCACTACACGT;R-primer:GCCAGAAGTCAGATGCTCAAGG);
7)菌检正确的克隆,命名为F2202-NKI451-VT,转接至4mL含有Kana抗性的液体培养基中,37℃、225rpm培养16-18h,然后进行质粒抽提,做酶切验证;
2、制备用于注射的质粒:将步骤(1)的载体所得载体转化大肠杆菌,经在37℃、225rpm培养16-18h后提取质粒,经测序验证后得到F2202-NKI451-VT质粒(如图7和8)。
3、按照现有技术获取雌鼠受精卵;
4、Donor/Cas9蛋白/gRNA共注射:
将质粒和Cas9蛋白、gRNA共同注射至受精卵核区;将注射后的受精卵移植到代孕鼠输卵管,等小鼠出生;
5、F0+F1小鼠鉴定:对F0小鼠PCR鉴定/测序,其中阳性克隆送测验证 arm/KI接口;然后使F0阳性小鼠与WT小鼠交配,繁育F1小鼠;对F1小鼠 PCR鉴定/测序(其中,引物1Fprimer:5’-GTACATCCACA GCATCTTCCAAG- 3,R primer:5’-GATGGGGAGAGTGAAGCAGAACG-3;引物2F primer:5’- GCATCTGACTTCTGGCTAATAAAG-3,R primer:5’-GCCTTGACCTAAGAGATGATGCGAC-3)
结果如图9所示,F1 7、8、11和12为基因阳性表达小鼠;再对PCR/测序验证后的阳性小鼠进行Southern检测,结果如图10所示,F1 7、8、11和12确认为基因阳性表达小鼠。
二、H11-MIR29A-cKI小鼠的应用
H11-MIR29A-cKI小鼠成功制备后可与特异性白蛋白(Albumin)基因启动子驱动的Cre重组酶转基因小鼠(Alb-Cre+)杂交,如图11所示,得到mir-29a特异性高表达的小鼠,获得深入研究mir-29a基因功能的动物模型,命名为“miR-29a 条件性敲入小鼠”。
miR-29a条件性敲入小鼠可作为研究原发性肝细胞癌和慢性血吸虫感染或晚期血吸虫病引起的肝纤维化的动物模型,为后续进一步研究肝细胞癌和血吸虫病引起的肝纤维化药物提供依据和基础。
序列表
<110> 华中科技大学同济医学院附属同济医院
<120> mir-29a基因在检测肝癌及肝纤维化中的应用及所述基因条件敲入小鼠的构建方法
<160> 13
<170> SIPOSequenceListing 1.0
<210> 13
<211> 664
<212> DNA
<213> 未知(Unknown)
<400> 13
gatgtgtgta ggttgtatgc aaatactaca ccattttcta tcagagactt gagcatctgt 60
ggattttggt atccaagggg ctttctggaa ccaatccctc aaggatacca agggatgaat 120
gtaattgtac aggatatcgc attgttggaa ttttatactt ctttgtggaa taaacctata 180
gcacttaata gatagtacag actcattcca ttgtgcctgg gttaaagagc ccaatgtatg 240
ctggatttag taagatttgg gccctcccaa ccctcacgac cttctgtgac cccttagagg 300
atgactgatt tcttttggtg ttcagagtca atataatttt ctagcaccat ctgaaatcgg 360
ttataatgat tggggaagag caccatgatg ctgactgctg agaggaaatg tattggtgac 420
cgttggggcc atggacaaga actaagaaaa caaatgcaaa gcaataatgc aaaggtgatt 480
tttcttcttc cagtttctaa gttgaatttc actgacctga attgcatgtg gtataatact 540
aacaaatggt tcactattag catatcatga atggttatac tttatagaaa ttccatagac 600
ttggtggggg ttttgttttg gtgacggata cctagaaaca ctcctgggga aaatcgatga 660
ctgg 664
<210> 1
<211> 19
<212> DNA
<213> 未知(Unknown)
<400> 1
cgccatcaag gtctactgc 19
<210> 2
<211> 18
<212> DNA
<213> 未知(Unknown)
<400> 2
acgggaatcc atcggtca 18
<210> 3
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 3
gcccacagcc ttctacacct 20
<210> 4
<211> 19
<212> DNA
<213> 未知(Unknown)
<400> 4
gccagggtca ccatttctc 19
<210> 5
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 5
gaagtatccg atagaacacg 20
<210> 6
<211> 20
<212> DNA
<213> 未知(Unknown)
<400> 6
ctcaaacata atatgggtca 20
<210> 7
<211> 22
<212> DNA
<213> 未知(Unknown)
<400> 7
tgacgtcact gggggttgta cc 22
<210> 8
<211> 21
<212> DNA
<213> 未知(Unknown)
<400> 8
ggttcatgtc atggatggtg c 21
<210> 9
<211> 19
<212> DNA
<213> 未知(Unknown)
<400> 9
cggatttggt cgtattggg 19
<210> 10
<211> 18
<212> DNA
<213> 未知(Unknown)
<400> 10
ctcgctcctg gaagatgg 18
<210> 11
<211> 26
<212> DNA
<213> 未知(Unknown)
<400> 11
ttaagggttc cggatccact acacgt 26
<210> 12
<211> 22
<212> DNA
<213> 未知(Unknown)
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gccagaagtc agatgctcaa gg 22
Claims (4)
1.mir-29a基因在检测原发性肝细胞癌上的应用。
2.mir-29a基因在检测日本血吸虫肝纤维化上的应用。
3.mir-29a基因条件敲入小鼠的构建方法,所述方法包括以下步骤:
(1)、构建human MIR29A-H11-cKI donor载体
(a)合成mir-29a基因序列,如SEQ No.1所示;
(b)构建H11位点基础骨架,使用AsiSI/PacI双酶切VB027载体骨架,胶回收11.5kb片段;
(c)连接片段的获取:使用EcoRI酶切步骤(a)的Mir-29基因合成产物合成质粒,胶回收片段;
(d)回收所得片段和骨架使用In-fusion连接酶进行连接;
(e)连接产物转化后进行培养,所得培养物中挑选单菌落进行菌落PCR鉴定;
(f)将PCR检验正确的克隆命名为F2202-NKI451-VT,转接至4mL含有Kana抗性的液体培养基中,37℃、225rpm培养16-18h,质粒抽提后进行做酶切验证,得到含mir-29a基因的载体;
(2)、制备质粒:
将步骤(1)的所得载体转化大肠杆菌,经在37℃、225rpm培养16-18h后提取质粒,经测序验证后得到F2202-NKI451-VT质粒;
(3)、获取雌鼠受精卵,将步骤(2)所得质粒和Cas9蛋白、gRNA共同移至受精卵核区,然后由代孕鼠代孕;
(4)、F0+F1小鼠鉴定:小鼠出生后,对F0小鼠PCR鉴定/测序,其中阳性克隆验证正确后,使F0阳性小鼠与WT小鼠交配,繁育F1小鼠,测序正确的F1小鼠即为mir-29a基因条件敲入小鼠。
4.根据权利要求3所述的构建方法得到的mir-29a基因条件敲入小鼠在研究原发性肝细胞癌和/或日本血吸虫肝纤维化上的应用。
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