CN105463027A - 一种高肌肉量及肥厚型心肌病模型克隆猪的制备方法 - Google Patents
一种高肌肉量及肥厚型心肌病模型克隆猪的制备方法 Download PDFInfo
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Abstract
本发明提供了Trim63基因在制备高肌肉量及肥厚型心肌病模型克隆猪中的应用。将修饰猪Trim63基因的体细胞作为核移植供体细胞,卵母细胞为核移植受体细胞,通过体细胞核移植技术获得克隆胚胎;将克隆胚胎移入猪子宫内妊娠即可获得Trim63基因修饰后的高肌肉量及肥厚型心肌病模型克隆猪,无需再对克隆猪进行手术等人工干预手段,提高了构建疾病模型的效率。本发明还首次在大动物上利用成对Cas9n打靶载体对大动物进行基因编辑,该方法成本低,大幅缩短获得纯合子猪的时间,为大动物利用CRISPR/Cas9技术进行基因功能研究及疾病模型建立奠定了基础。
Description
技术领域
本发明涉及基因工程领域,具体地说,涉及一种利用CRISPR/Cas9系统及双Cas9n技术进行的Trim63基因的修饰。
背景技术
肌肉由肌纤维组成,动物在出生后肌纤维的数量基本保持不变,因此肌肉大小主要取决于肌纤维的大小。而肌纤维的大小取决于肌纤维细胞内蛋白质的合成与降解的平衡:当蛋白质合成大于降解时,肌纤维表现为肥大,反之当蛋白质降解大于合成时,肌纤维则表现为萎缩。
作为肌肉特异表达的E3泛素连接酶,三结构域蛋白63(tripartitemotif-containing63,Trim63)和F-box蛋白32(F-boxprotein32,Fbxo32)调控了绝大多数肌浆蛋白和肌肉生长调控因子的降解。Trim63和Fbxo32于2001年被发现在几乎所有的肌肉萎缩的模型中都明显上调,并且在人和大鼠中都只在骨骼肌和心脏中表达。在Trim63和Fbxo32敲除后,小鼠都表现出显著的抗肌肉萎缩的能力,并且在生长和繁殖方面表现完全正常。但是对于敲除TRIM63后肌肉量变化并无系统研究。
有研究表明,小鼠双等位基因敲除Trim63和Trim55之后,能够表现自发的心肌病。但是当小鼠的四个等位基因中的一个尚未缺失时(Trim63-/-//trim55+/-或Trim63+/-,Trim55-/-),敲除鼠与野生鼠无异(WillisMSetal.Muscleringfinger1andmuscleringfinger2arenecessarybutfunctionallyredundantduringdevelopmentalcardiacgrowthandregulateE2F1-mediatedgeneexpressioninvivo.CellBiochemFunct.2014Jan;32(1):39-50.doi:10.1002/cbf.2969.Epub2013Mar20.)。而单独缺失Trim63双等位基因的小鼠,需经过主动脉缩窄术才能诱导出心肌肥大的症状,若未经手术,则无法出现Trim63-/-鼠的病理表型(WillisMS,IkeC,LiL,etal.Muscleringfinger1,butnotmuscleringfinger2,regulatescardiachypertrophyinvivo.CircRes.2007;100:456–9.)。这与人类发生肥厚型心肌病的机理有极大的不同。
而对于猪等大动物,目前尚未有Trim63基因对于肌肉量的维持以及肥厚型心肌病的功能方面的研究。
猪作为主要家畜之一,由于存在肉类市场方面的需求,需要制备高肌肉量克隆猪。而猪的心脏结构与人类几乎一模一样,是人类心血管研究中最重要的一种模式生物,构建肥厚型心肌病疾病模型有助于人类加深对该类疾病的认识,从而提供更多更好的治疗方案。因此,亟需一种能够获得该模型克隆猪的方法。
发明内容
为了解决现有技术中存在的问题,本发明的目的是提供Trim63基因在制备高肌肉量及肥厚型心肌病模型克隆猪中的应用以及一种高肌肉量及肥厚型心肌病表型的克隆猪的制备方法。
为了实现本发明目的,本发明的技术方案如下:
第一方面,本发明提供Trim63基因在制备高肌肉量及肥厚型心肌病模型克隆猪中的应用。
所述应用为将修饰猪Trim63基因的体细胞作为核移植供体细胞,卵母细胞为核移植受体细胞,通过体细胞核移植技术获得克隆胚胎;将克隆胚胎移入猪子宫内妊娠即可获得Trim63基因修饰后的高肌肉量及肥厚型心肌病模型克隆猪,无需再对克隆猪进行主动脉缩窄术等人工干预手段。
第二方面,本发明提供一种高肌肉量及肥厚型心肌病表型的克隆猪的制备方法,所述方法包括如下步骤:
1)根据特异性靶向Trim63基因第一外显子的sgRNA,构建针对Trim63基因第一外显子靶点的打靶载体;所述猪Trim63基因第1外显子的核苷酸序列如SEQIDNO.1所示,所述sgRNA的DNA序列如SEQIDNO.2和/或SEQIDNO.3所示;
2)将所述打靶载体转入猪成纤维细胞,获得阳性细胞克隆;以阳性细胞为核移植供体细胞,卵母细胞为核移植受体细胞,通过体细胞核移植技术获得克隆胚胎;将克隆胚胎移入猪子宫内妊娠获得Trim63基因修饰后的高肌肉量及肥厚型心肌病模型克隆猪。
可选的,所述打靶载体为CRISPR/Cas9打靶载体或成对Cas9n打靶载体。优选使用成对Cas9n打靶载体,其能更大面积的造成Trim63基因第一外显子的插入突变,使该基因发生移码突变,造成终止密码子提前出现于第一外显子,从而翻译出一个截短的突变蛋白,达到基因敲除的目的。
当所述打靶载体为CRISPR/Cas9打靶载体时:
所述CRISPR/Cas9打靶载体的构建方法为:
1)将SEQIDNO.4和SEQIDNO.5所示序列进行互补配对,形成双链DNA;
2)将px330骨架载体用限制性内切酶BbsⅠ进行酶切过夜,回收后,与步骤1)得到的双链DNA连接,即得;
或:
1)将SEQIDNO.6和SEQIDNO.7所示序列进行互补配对,形成双链DNA;
2)将px330骨架载体用限制性内切酶BbsⅠ进行酶切过夜,回收后,与步骤1)得到的双链DNA连接,即得。
当所述打靶载体为成对Cas9n打靶载体时,需按物质的量1:1的比例混合后,转入猪成纤维细胞。
所述成对Cas9n打靶载体的构建方法为:
1)将SEQIDNO.4和SEQIDNO.5所示序列进行互补配对,形成双链DNA;
2)将SEQIDNO.6和SEQIDNO.7所示序列进行互补配对,形成双链DNA;
3)将px335骨架载体用限制性内切酶BbsⅠ进行酶切过夜,回收后,分别与步骤1)和步骤2)得到的双链DNA连接,即得。
其中,所述px330骨架载体和px335骨架载体为本领域常规载体,可购自Addgene公司。
作为优选,本发明提供一个最佳的互补配对反应程序,具体为:94℃变性5min,35℃退火10min,0~4℃保存。可选的,可将反应物放置在冰上进行保存。
进一步地,所述打靶载体转入猪成纤维细胞的方法为,将打靶载体用电击转染或脂质体转染的方法转入猪成纤维细胞。
所述打靶载体转入猪成纤维细胞的比例为:每1×106个猪成纤维细胞转入打靶载体4-6μg。
本发明的有益效果在于:
本发明提供了Trim63基因在制备高肌肉量及肥厚型心肌病模型克隆猪中的应用。将修饰猪Trim63基因的体细胞作为核移植供体细胞,卵母细胞为核移植受体细胞,通过体细胞核移植技术获得克隆胚胎;将克隆胚胎移入猪子宫内妊娠即可获得Trim63基因修饰后的高肌肉量及肥厚型心肌病模型克隆猪,无需再对克隆猪进行主动脉缩窄术等人工干预手段,提高了构建疾病模型的便利性。
本发明还首次在大动物上利用成对Cas9n打靶载体对大动物进行基因编辑,该方法成本低,大幅缩短获得纯合子猪的时间,为大动物利用CRISPR/Cas9技术进行基因功能研究及疾病模型建立奠定了基础。
附图说明
图1是本发明实施例1中T7E1酶切法鉴定在猪胚胎成纤维细胞上px330质粒对基因组的切割情况,pM1和pM9分别表示本发明获得的两个px330质粒。
图2是本发明实施例2中T7E1酶切法鉴定在猪胚胎成纤维细胞上成对px335质粒对基因组的切割情况,pM1n和pM9n分别表示本发明获得的两个px335质粒。
图3是本发明实例4中,针对克隆猪Trim63基因第1外显子插入的83bp的测序峰图。峰图单一无明显杂峰,说明克隆猪Trim63基因为纯合突变。
具体实施方式
下面将结合实施例对本发明的优选实施方式进行详细说明。需要理解的是以下实施例的给出仅是为了起到说明的目的,并不是用于对本发明的范围进行限制。本领域的技术人员在不背离本发明的宗旨和精神的情况下,可以对本发明进行各种修改和替换。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
其中:px330载体、px335载体购于Addgene公司;T4DNA连接酶、限制性内切酶购于大连TaKaRa公司;引物合成及序列测定由深圳华大和北京美吉完成;KODDNA聚合酶购于上海东洋纺公司;T7E1酶购于NEB公司;质粒去内毒素大提试剂盒、胶回收试剂盒购于Omega公司;基因组提取试剂盒购于QIAGEN公司;酶切、连接、回收、转化、PCR扩增等常规实验操作步骤详见《分子克隆(第三版)》。
实施例1CRISPR-Cas9打靶载体的构建
1、利用张锋实验室网站(http://crispr.genome-engineering.org/)对猪Trim63基因第1外显子的打靶位点进行预测。根据自我评估和预测结果中的评分,从候选的靶位点中选择两个,命名为pM1、pM9,其sgRNA序列分别为TGGGAACCCCATGGAGAACC(SEQIDNO.2)和TGACTTATAATCCATATTGT(SEQIDNO.3)。根据sgRNA序列合成互补配对的寡聚核苷酸,如表1所示,其中小写字母为酶切位点。
表1寡聚核苷酸序列
名称 | 序列(5’-3’) |
pM1F | caccgTGGGAACCCCATGGAGAACC(SEQ ID NO.4) |
pM1R | aaacGGTTCTCCATGGGGTTCCCAc(SEQ ID NO.5) |
pM9F | caccgTGACTTATAATCCATATTGT(SEQ ID NO.6) |
pM9R | aaacACAATATGGATTATAAGTCAc(SEQ ID NO.7) |
2、共构建2个打靶载体,命名为px330-pM1、px330-pM9,分别使用表1的两对寡聚核苷酸,构建过程如下:94℃,5min,再35℃,10min,然后立即放冰上。px330骨架载体用限制性内切酶BbsⅠ进行酶切过夜,回收后,与退火的寡聚核苷酸16℃连接3h。通过常规转化法进行转化、涂板。待单菌落长成后,挑取数个扩大培养并测序。测序验证正确,说明本发明成功构建两个CRISPR-Cas9打靶载体。
3、阳性单菌落扩大培养
具体步骤为:a.初始培养,用枪头挑取阳性单菌落,加入盛有5mlLB培养基(胰蛋白胨10g、酵母提取物5g、NaCl10g溶于1L蒸馏水中)的灭菌管中,37℃,220rpm,培养8h至12h;b.扩大培养,将过夜培养液按体积1:500的比例转移到盛有100mlLB培养基的灭菌三角瓶中,37℃,220rpm,培养12h至16h;
4、px330质粒去内毒素大提
按照质粒去内毒素大提试剂盒(EndoFreePlasmidMaxiKit)上提供的方法,提取px330质粒,所提的质粒用于细胞的转染。
5、细胞转染
细胞转染采用LonzaNucleofector进行电转。具体流程如下:a.将消化并收集的6孔细胞培养板中一个孔的猪成纤维细胞(约1×106个)、4μgpx330质粒和100μlNucleofector试剂混匀,装入电击杯用T-016程序进行电击转染;b.电击结束后,沿电击杯内壁缓慢加入37℃预热的成纤维细胞培养基(10%FBS+DMEM)500μL,将细胞接种于6孔细胞培养板的一个孔内;c.用无筛选药物的成纤维细胞培养基(10%FBS+DMEM)于37.5℃,5%CO2培养箱培养。
6、打靶效率的检测
按照基因组提取试剂盒(DneasyBlood&TissueKit)上提供的方法,提取细胞基因组。以提取的基因组和野生型猪成纤维细胞基因组为模板,用KODDNA聚合酶进行PCR,扩增出629bp的片段,引物为t1a-F5’-TCTTTCAGGCTTGGAGGAAA-3’t1a-R5’-ATTCCTTCTCCATGACGCAC-3’。扩增条件为94℃,2min;94℃,30sec;60℃,30sec;68℃,40sec;68℃,7min;35个循环,1.0%琼脂糖电泳观察结果,然后回收PCR产物,测浓度。取400ngPCR回收产物进行退火,从95℃程序降温至4℃。退火后的产物用T7E1酶进行酶切,37℃1h,体系为:退火产物10μl,NEBbuffer22μl,T7E10.5μl,ddH2O补足至20μl。酶切完成后用PAGE胶电泳观察结果,两个px330质粒均能在猪成纤维细胞上发挥切割基因组的作用,如图1所示。
实施例2Cas9n打靶载体的构建
1、共构建2个打靶载体,命名为px335-pM1n、px335-pM9n,分别使用表1的两对寡聚核苷酸,构建过程如下:94℃,5min,再35℃,10min,然后立即放冰上,对寡聚核苷酸进行退火。px335骨架载体用限制性内切酶BbsⅠ进行酶切过夜,回收后,与退火的寡聚核苷酸16℃连接3h。通过常规转化法进行转化、涂板。待单菌落长成后,挑取数个扩大培养并测序。测序验证正确,说明本发明成功构建两个Cas9n打靶载体。
3、阳性单菌落扩大培养
具体步骤为:a.初始培养,用枪头挑取阳性单菌落,加入盛有5mlLB培养基(胰蛋白胨10g、酵母提取物5g、NaCl10g溶于1L蒸馏水中)的灭菌管中,37℃,220rpm,培养8h至12h;b.扩大培养,将过夜培养液按体积1:500的比例转移到盛有100mlLB培养基的灭菌三角瓶中,37℃,220rpm,培养12h至16h;
4、px335质粒去内毒素大提
按照质粒去内毒素大提试剂盒(EndoFreePlasmidMaxiKit)上提供的方法,提取px335质粒,所提的质粒用于细胞的转染。
5、细胞转染
细胞转染采用LonzaNucleofector进行电转。具体流程如下:a.将消化并收集的6孔细胞培养板中一个孔的猪成纤维细胞(约1×106个)、一对px335质粒各2μg和100μlNucleofector试剂混匀,装入电击杯用T-016程序进行电击转染;b.电击结束后,沿电击杯内壁缓慢加入37℃预热的成纤维细胞培养基(10%FBS+DMEM)500μL,将细胞接种于6孔细胞培养板的一个孔内;c.用无筛选药物的成纤维细胞培养基(10%FBS+DMEM)于37.5℃,5%CO2培养箱培养。
6、打靶效率的检测
按照基因组提取试剂盒(DneasyBlood&TissueKit)上提供的方法,提取细胞基因组。以提取的基因组和野生型猪成纤维细胞基因组为模板,用KODDNA聚合酶进行PCR,扩增出629bp的片段,引物为t1a-F5’-TCTTTCAGGCTTGGAGGAAA-3’;t1a-R5’-ATTCCTTCTCCATGACGCAC-3’。扩增条件为94℃,2min;94℃,30sec;60℃,30sec;68℃,40sec;68℃,7min;35个循环。1.0%琼脂糖电泳观察结果,然后回收PCR产物,测浓度。取400ngPCR回收产物进行退火,从95℃程序降温至4℃。退火后的产物用T7E1酶进行酶切,37℃1h,体系为:退火产物10μl,NEBbuffer22μl,T7E10.5μl,ddH2O补足至20μl。酶切完成后用PAGE胶电泳观察结果,成对Cas9n能在猪成纤维细胞上发挥切割基因组的作用,如图2所示。
实施例3阳性细胞单克隆的筛选及鉴定
1、阳性单克隆细胞的筛选
消化并收集6孔细胞培养板中一个孔的猪成纤维细胞(约1×106个),将实施例2构建的打靶载体px335-pM1n和px335-pM9n按物质的量1:1的比例混合,取总质量4μg,按实施例2中的步骤5的方法进行转染后,放至CO2培养箱中,37.5℃培养。48h后细胞汇合度达到80-90%,此时将1个孔的细胞平均分到8个10cm培养皿中。24h后细胞贴壁,将培养基更换为含G418(600μg/mL)的成纤维细胞培养基(10%FBS+DMEM),每3~4d换一次液,培养基仍然为含G418(600μg/mL)的成纤维细胞培养基。细胞培养6~9d后,可以观察到细胞克隆点形成。在显微镜下找到抗性细胞克隆点,用Marker笔做标记,倒掉培养基,PBS溶液清洗一次,用细胞克隆环将抗性细胞克隆点罩住,加入10~30μL在37℃预热的0.25%胰蛋白酶消化液,37.5℃消化细胞2min左右,加入细胞培养基终止消化反应,将消化下来的细胞接种到48孔细胞培养板中培养。待细胞汇合度达到90%时,消化细胞,接种到12孔细胞培养板中继续培养,原来48孔细胞培养板中的未被消化下来的细胞也继续培养以供提取细胞基因组DNA,细胞继续扩大培养至6孔细胞培养板,按照猪胚胎成纤维细胞冻存方法进行抗性细胞冻存。
2、阳性细胞单克隆的鉴定
对所挑取的34个细胞单克隆进行鉴定:
以提取的细胞单克隆基因组DNA为模板,用KODDNA聚合酶进行PCR,扩增出629bp的片段,引物为:
t1a-F5’-TCTTTCAGGCTTGGAGGAAA-3’;
t1a-R5’-ATTCCTTCTCCATGACGCAC-3’。
以野生型猪成纤维细胞基因组DNA为模板的PCR反应为阴性对照。扩增条件:94℃,2min;94℃,30sec;60℃,30sec;68℃,40sec;68℃,7min;35个循环,再加入1μLtaqDNA聚合酶,68℃,10min。扩增完成后,1.0%琼脂糖电泳观察结果。切胶回收PCR产物,测浓度。纯化后的PCR产物连入T载进行转化,阳性菌落进行测序,方能得到细胞单克隆的Trim63基因第1外显子序列。
本实施例中,鉴定结果说明阳性单克隆中Trim63基因第1外显子发生纯合83bp的插入。
实施例4Trim63基因修饰的高肌肉量及肥厚型心肌病模型克隆猪的制备与鉴定
1、Trim63基因修饰高肌肉量及肥厚型心肌病模型克隆猪的制备
以实施例3获得的阳性细胞为核移植供体细胞,以体外成熟的初情期前母猪卵母细胞为核移植受体细胞,将核移植供体细胞移入去核的卵母细胞,经电融合与激活,构建成克隆胚胎,挑选形态优良的克隆胚胎用手术法移入自然发情的经产母猪子宫内进行妊娠,手术法胚胎移植步骤为舒泰常规麻醉,在手术架上仰卧保定,腹中线做一个长约8cm的手术切口,曝露卵巢、输卵管及子宫,用胚胎移植管沿输卵管伞部进入约5cm,将胚胎(300枚以上)移植到输卵管壶腹部-峡部结合处。胚胎移植后30天B型超声波检测妊娠与否。
2、新生猪的PCR检测
对步骤1中妊娠足月出生的克隆猪进行PCR检测,检测方法与实施例3中细胞单克隆的检测方法一致。Trim63第一外显子测序结果如SEQIDNo.8所示,峰图如图3所示无杂峰,说明新生猪为纯合子。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
Claims (10)
1.Trim63基因在制备高肌肉量及肥厚型心肌病模型克隆猪中的应用。
2.根据权利要求1所述的应用,其特征在于,将修饰猪Trim63基因的体细胞作为核移植供体细胞,卵母细胞为核移植受体细胞,通过体细胞核移植技术获得克隆胚胎;将克隆胚胎移入猪子宫内妊娠获得Trim63基因修饰后的高肌肉量及肥厚型心肌病模型克隆猪。
3.一种高肌肉量及肥厚型心肌病表型的克隆猪的制备方法,其特征在于,所述方法包括如下步骤:
1)根据特异性靶向Trim63基因第一外显子的sgRNA,构建针对Trim63基因第一外显子靶点的打靶载体;所述sgRNA的DNA序列如SEQIDNO.2和/或SEQIDNO.3所示;
2)将所述打靶载体转入猪成纤维细胞,获得阳性细胞克隆;以阳性细胞为核移植供体细胞,卵母细胞为核移植受体细胞,通过体细胞核移植技术获得克隆胚胎;将克隆胚胎移入猪子宫内妊娠获得Trim63基因修饰后的高肌肉量及肥厚型心肌病模型克隆猪。
4.根据权利要求3所述的方法,其特征在于,所述打靶载体为CRISPR/Cas9打靶载体或成对Cas9n打靶载体。
5.根据权利要求4所述的方法,其特征在于,所述打靶载体为CRISPR/Cas9打靶载体时:
所述CRISPR/Cas9打靶载体的构建方法为:
1)将SEQIDNO.4和SEQIDNO.5所示序列进行互补配对,形成双链DNA;
2)将px330骨架载体用限制性内切酶BbsⅠ进行酶切过夜,回收后,与步骤1)得到的双链DNA连接,即得;
或:
1)将SEQIDNO.6和SEQIDNO.7所示序列进行互补配对,形成双链DNA;
2)将px330骨架载体用限制性内切酶BbsⅠ进行酶切过夜,回收后,与步骤1)得到的双链DNA连接,即得。
6.根据权利要求4所述的方法,其特征在于,所述打靶载体为成对Cas9n打靶载体时,需按物质的量1:1的比例混合后,转入猪成纤维细胞。
7.根据权利要求6所述的方法,其特征在于,所述成对Cas9n打靶载体的构建方法为:
1)将SEQIDNO.4和SEQIDNO.5所示序列进行互补配对,形成双链DNA;
2)将SEQIDNO.6和SEQIDNO.7所示序列进行互补配对,形成双链DNA;
3)将px335骨架载体用限制性内切酶BbsⅠ进行酶切过夜,回收后,分别与步骤1)和步骤2)得到的双链DNA连接,即得。
8.根据权利要求5或7所述的方法,其特征在于,所述互补配对的反应程序为:94℃变性5min,35℃退火10min,0~4℃保存。
9.根据权利要求3所述的方法,其特征在于,将打靶载体用电击转染或脂质体转染的方法转入猪成纤维细胞。
10.根据权利要求9所述的方法,其特征在于,所述打靶载体转入猪成纤维细胞的比例为:每1×106个猪成纤维细胞转入打靶载体4-6μg。
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