CN112715483B - 一种制备使心功能降低的突变型CNPase斑马鱼模型及应用的方法 - Google Patents
一种制备使心功能降低的突变型CNPase斑马鱼模型及应用的方法 Download PDFInfo
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Abstract
本发明涉及到了CNPase突变型斑马鱼的制备，并通过确认了该突变体模型可自发严重心衰伴随的心率过缓乃心力衰竭。本发明采用CRISPR‑Cas9技术结合显微注射技术，特异性敲除CNPase，制备CNPase突变型F1代，并通过桑格测序的方法进行确认；通过杂交以及筛选，制备出心脏特异性标记绿色荧光的斑马鱼纯合突变品系，通过心脏病理学以及动力学指标，确认了CNPase纯合突变型斑马鱼可自发心肌肥大，部分发展至心力衰竭。本发明制备了CNPase突变型斑马鱼，为了研究CNPase的功能提供了动物模型，特别是为严重心衰伴随的心律过缓相关疾病的研究提供了极大的便利。
Description
背景技术
2’,3’-环核苷酸-3’-磷酸二酯酶(2',3'-Cyclic-nucleotide 3'-phosphodiesterase，CNPase)，具有催化2’，3’-cAMP和2’，3’-cGMP降解的功能。在本团队的专利申请书(申请号)中，使用腺相关病毒重组载体在腹主动脉结扎的大鼠心脏中过表达CNPase，该方法对心肌肥大疾病具有良好的治疗作用。目前CNPase突变型小鼠已有报道，但目前CNPase突变型斑马鱼相关的动物模型尚无报道，而且与斑马鱼心脏功能的研究尚未见报道。
本发明提供例如CNPase突变型斑马鱼的制备方法，可为CNPase在心脏中的功能研究提供动物模型。斑马鱼个体小，繁殖代际时间短，养殖成本低且易于管理；斑马鱼卵透明，便于观察和记录以及操作，其中myl7:GFP品系的斑马鱼是心肌特异性标记绿色荧光的斑马鱼品系，本发明采用该品系用于制备CNPase突变型斑马鱼。
Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)-Cas9是古细菌防御噬菌体入侵的一套分子免疫系统，具有识别外源性和切割外源性DNA的功能。其中CRISPR是在细菌中发现的有规律成簇又带间隔的短回文序列，Cas9是。CRISPR-Cas9被用于基因组的精确编辑，具有高效，方便，快捷的优点，在本发明中，CRISPR-Cas9结合显微注射技术用于在斑马鱼CNPase基因的敲除。
本发明涉及到了CNPase突变型动物模型的建立以及在心血管领域的研究以及应用。
发明内容
本发明深入研究了CNPase与严重心衰伴随的心律过缓类疾病的联系，证实了CNPase是心功能维持所必不可少的，是潜在抗心衰的靶点。为了验证该猜想，发明人选用myl7：GFP斑马鱼胚胎作为验证心脏功能的模型。myl7：GFP斑马鱼为心肌特异性表达GFP蛋白的品系，而斑马鱼胚胎具有通体透明的特点，方便在胎胚时期读心功能以及血流动力学相关指标进行检测。采用吗啉环寡聚核苷酸(morpholino)和CRISPR/Cas9介导的cnpase基因编辑系统，通过显微注射的方式，对CNPase进行敲除和敲降。心脏形态以及动力学方面的评价结果显示，CNPase敲除或敲降可诱导斑马鱼自发性的严重心衰伴随的心律过缓乃至心衰，这证明CNPase是心脏功能维持所必不可少的，为CNPase作为治疗心肌病靶点提供了理论基础。
本发明描述了一种制备CNPase突变型斑马鱼的方法，并描述了CNPase敲除后对斑马鱼心脏功能的影响，其特征在于，制备方法如下：
(1)针对CNPase的第一个外显子序列，设计特异性的一对sgRNA序列，sgRNA1其序列特征为sgRNA2其序列特征为/>其中斜体序列为T7启动子序列，红色部分为sgRNA核心序列；通用引物的序列为5′-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3′。
(2)采用fill-in PCR方法和纯化的方法制备sgRNA DNA模板，结合体外转录以及乙醇沉淀的方法，获取sgRNA。
(3)通过显微注射的方法，在斑马鱼胚胎单细胞期，注射1nL sgRNA与Cas9蛋白混合物；培育F1代。
(4)F1通过提取鱼尾DNA，使用PCR扩增，采用桑格测序法筛选和确认CNPase突变型斑马鱼；采用突变型F1斑马鱼进行交配，筛选F2代斑马鱼。
CNPase突变型斑马鱼，其病理特征在于：心脏离心性扩脏性肥大，出现围心腔水肿；心率减缓，由对照组的约160次/min下降为约80次/min；心输出量(cardiac output)下降大约50％左右；收缩分数(fractional shortening)以及射血分数(ejection fraction)大约下降50％左右；部分突变型斑马鱼可自发心力衰竭。
本发明CNPase突变型斑马鱼可用于CNPase对心脏功能调控的研究，也可以用作自发性严重心衰伴随的心律过缓和心力衰竭的动物模型，用于药物筛选等用途。
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例的详细说明如后。
具体实施方式
为了证明CNPase对斑马鱼心脏功能维持必不可少，选择在斑马鱼胚胎1～2细胞期，采用显微注射吗啡啉反义寡核苷酸(morpholino antisense oligonucleotides,MO)或使用CRISPR/Cas9介导的cnpase基因编辑系统通过显微注射方法敲降或敲除斑马鱼cnpase基因。在体视显微镜下观察受精后72-120h斑马鱼胚胎心脏,分析各时间点斑马鱼心脏环化情况。通过计数及测量各组斑马鱼胚胎受精后96h及120h时心室收缩分数(ventricularshortening fraction,VSF)，评价cnpase基因表达下调对斑马鱼心脏功能的影响。
本发明中列举的案例用于支持本发明的实验方法和结果，并验证本发明中使用的动物模型。本发明的所有实验均采用了适当的对照和统计检验。提供以下实施例来说明而非限制本发明。这些例子说明了一种制备严重心衰伴随心律过缓疾病的CNPase突变型斑马鱼模型及应用。
实验方法
斑马鱼与人类基因同源性较高，可以反映动物的新陈代谢和整体生理过程，且其心血管系统的早期发育过程与人类极为相似，其心血管系统有缺陷的突变体仍然可以长时间存活，这种现象为心血管发育遗传学研究提供了极为有利的条件。为了探索CNPase在斑马鱼中的功能，本项目采用吗啉代反义寡核苷酸(Morpholino，MO)技术，构建了抑制剪接的MO(MO-Spl)下调斑马鱼cnpase基因所需的重组质粒-cnpase-MO-Spl。通过显微注射的方式，将cnpase-MO-Spl或control-MO注射到1-2个细胞阶段的胚胎,阻遏原始转录本的剪接。
本研究利用CRISPR/Cas9系统介导的基因组定向编辑，在斑马鱼的cnpase编码区诱导突变等位基因。具体操作流程如下，(1)使用ENSEMBL数据库获取CNPase基因的外显子序列信息，使用CRISPRscan.org预测靶向的sgRNA，并通过公司合成相应的引物，引物序列信息见权利要求1；(2)sgRNA1与sgRNA2分别与universal primer混合，使用KOD-plus试剂(TOYOBO公司，KOD-201)进行PCR扩增，操作流程如产品说明书，通过电泳以及胶回收获取sgRNA1与sgRNA2 DNA模板；(3)使用纯化的DNA模板，采用T7体外转录试剂盒(Riobio，C11001-1)操作说明书进行操作，37℃孵育6h，之后加入DNase继续孵育20min，以清除DNA模板；采用无水乙醇沉淀法获取纯化的sgRNA，使用nanoDROP2000测量RNA浓度，分装后冻存在-80℃；(4)采用显微注射的方法，将gRNA1以及gRNA2与Cas9蛋白(Novoprotein，Cas9Nuclease，E365-01A)注射到单细胞期转基因斑马鱼胚胎中。
用PCR和亚克隆技术检测两个靶点的indel诱导率，从F1代鱼后代中筛选出纯合子突变鱼。结果成功构建了cnpase基因敲除斑马鱼动物模型，且初步研究发现F1代斑马鱼在cnpase编码区诱导突变等位基因后，出现心脏无搏动、心房和心室增大、心包积液。饲养F1三个半月后，通过提取鱼尾DNA，使用PCR扩增，采用桑格测序法筛选和确认CNPase突变型斑马鱼；采用突变型F1斑马鱼进行交配，筛选F2代斑马鱼。
实施例1
本案例主要说明一种制备严重心衰伴随心律过缓疾病的CNPase突变型斑马鱼模型。采用显微注射吗啡啉反义寡核苷酸(morpholino antisense oligonucleotides,MO)显微注射方法下调斑马鱼cnpase基因，以标准对照吗啡啉反义寡核苷酸(control-MO-Spl)注射组为对照。体视显微镜下观察各组斑马鱼胚胎发育情况,统计分析不同注射浓度斑马鱼胚胎死亡以及畸形数量。
表1.高浓度cnpase-MO敲除后斑马鱼与对照组胚胎的存活率(％)。
| 存活率(％) | control-MO-Spl | cnpase-MO-Spl |
| 24h | 100 | 100 |
| 48h | 84.21 | 14.65 |
| 72h | 63.16 | 9.48 |
| 96h | 63.16 | 9.48 |
| 120h | 63.16 | 9.48 |
实施例2
为了确保存活的胚胎数量，我们随后将cnpase-MO-Spl的使用量降低至1-2nL。注射cnpase-MO-Spl的第4天，发现斑马鱼胚胎早期出现了心脏积液及体节发育异常并导致体轴弯曲。与control-MO相比，cnpase-MO-Spl斑马鱼的心房和心室相对较大。此外，心功能评价指标的结果显示，cnpase-MO-Spl斑马鱼的心跳、心输出量、分数缩短和射血分数均下降，揭示敲降cnpase后，心脏无法将足够的富氧血液泵送到身体其它部位,提示敲降cnpase可诱导斑马鱼自发性的严重心衰伴随的心律过缓乃至心衰。
表2.cnpase-MO斑马鱼胚胎的心功能评价(n＝3-5)
| control-MO-Spl | cnpase-MO-Spl | |
| 心率(BPM) | 161.2±4.295 | 81.38±22.32 |
| 分数缩短(FS) | 28.36±2.992 | 16.76±1.464 |
| 左心室射血分数(EF) | 43.33±1.953 | 19.66±0.8568 |
| 心输出量(CO) | 71.37±7.423 | 32.35±9.038 |
实施例3
使用了CRISPR/Cas9介导的cnpase基因编辑系统的4天后，我们发现其表型与注射了cnpase-MO-Spl是一样的，斑马鱼胚胎早期均出现心脏积液及体节发育异常并导致体轴弯曲。与对照组比较cnpase-MO-Spl注射组斑马鱼胚胎心前区水肿、心脏环化异常、心脏搏动减弱、心室离心性肥大。在使用两种独立的、原理完全不同的敲降/敲除方法后，我们得到的敲降/敲除效果(表型)是一样的，确认了本项目建立的基因敲降方法是有效且特异的。
序列表
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Claims (7)
1.一种制备使心功能降低的突变型CNPase斑马鱼模型的方法，其特征在于，所述的方法包括：
(1)针对CNPase的第一个外显子序列，设计特异性的一对sgRNA序列，sgRNA1其序列特征为5′-TAATACGACTCACTATA[GGAAGTGGCGGCTCAGCAAGAGG]GTTTTAGAGCTAGAA-3′，sgRNA2其序列特征为TAATACGACTCACTATA[GGAGGAGGAAGCTGTGAAAGAGG]GTTTTAGAGCTAGAA-3′，其中斜体序列为T7启动子序列，红色部分为sgRNA核心序列；通用引物的序列为5′-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTG CTATTTCTAGCTCTAAAAC-3′；
(2)采用fill-in PCR方法和纯化的方法制备sgRNADNA模板，结合体外转录以及乙醇沉淀的方法，获取sgRNA；
(3)通过显微注射的方法，在斑马鱼胚胎单细胞期，注射1nL sgRNA与Cas9蛋白混合物；培育F1代；
(4)F1通过提取鱼尾DNA，使用PCR扩增，采用桑格测序法筛选和确认CNPase突变型斑马鱼；采用突变型F1斑马鱼进行交配，筛选F2代斑马鱼。
2.根据权利要求1所述的一种制备使心功能降低的突变型CNPase斑马鱼模型的方法，其特征在于，所述的心功能降低包括心力衰竭和心动过缓。
3.根据权利要求2所述的一种制备使心功能降低的突变型CNPase斑马鱼模型的方法，其特征在于，所述的心力衰竭在于伴随心律过缓，突变型CNPase斑马鱼出现心脏离心性扩脏性肥大、围心腔水肿及心率减缓。
4.根据权利要求3所述的一种制备使心功能降低的突变型CNPase斑马鱼模型的方法，其特征在于，所述的心率减缓包括心输出量下降大约50％，收缩分数和射血分数均大约下降50％，部分突变型斑马鱼自发心力衰竭。
5.根据权利要求1所述的一种制备使心功能降低的突变型CNPase斑马鱼模型的方法，其特征在于，所述的心功能降低还包括心律失常。
6.根据权利要求1所述的一种制备使心功能降低的突变型CNPase斑马鱼模型的方法，其特征在于，采用吗啉代反义寡核苷酸MO技术构建抑制剪接的MO-Spl下调斑马鱼cnpase基因所需的重组质粒-cnpase-MO-Spl。
7.根据权利要求1所述的一种制备使心功能降低的突变型CNPase斑马鱼模型的方法，其特征在于，通过显微注射的方式将cnpase-MO-Spl注射到1-2个细胞阶段的胚胎，阻遏原始转录本的剪接。
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