CN113584079A - 一种应用于钙离子成像的斑马鱼心脏特异标记品系的建立 - Google Patents
一种应用于钙离子成像的斑马鱼心脏特异标记品系的建立 Download PDFInfo
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Abstract
本发明涉及基因敲入技术领域,公开了一种应用于钙离子成像的斑马鱼心脏特异标记品系的构建方法。该方法使用了CRISPR/Cas9基因编辑技术,将编码钙离子浓度指示剂的GCaMP5G蛋白的基因编码序列敲入野生型斑马鱼myl7基因的7号外显子内。方法内容包括:确定将GCaMP5G基因序列敲入斑马鱼myl7基因的7号外显子的特异性靶位点以及相应sgRNA的合成,GCaMP5G基因敲入斑马鱼myl7特异位点的序列合成,GCaMP5G基因敲入斑马鱼品系筛选与鉴定。myl7基因的打靶序列如CRISPR/Cas9打靶***的流程图所示,GCaMP5G蛋白的基因编码序列如图1所示。本发明的斑马鱼品系有助于钙离子成像观察心脏形态发生的整个过程以及心脏收缩和舒张时引起的钙流变化,可以广泛用于心脏疾病的病理研究,并对药物筛选的体外验证提供了良好的基础。
Description
技术领域
本发明属于遗传学和生物技术领域,特别是公开了一种应用于钙离子成像的斑马鱼心脏特异标记品系的构建方法。
背景技术
钙离子成像技术(calcium imaging)是指利用钙离子指示剂监测组织内钙离子浓度的方法。想要对生物体内钙离子的动态变化进行有效的检测,钙离子指示剂的选择显得尤为重要。钙离子荧光指示剂在未结合钙离子前几乎无荧光,与钙离子结合后,荧光强度显著增强。利用这一原理,可以通过指示剂的信号强弱来观察细胞内钙离子浓度水平的变化。目前使用的钙离子指示剂有化学性钙离子指示剂(chemical indicators)和基因编码钙离子指示剂(genetically-encoded Ca2+ indicators,GECIs)两类,而GECIs由于其高灵敏度、高亮度和动态荧光范围等优点,已成为应用最为广泛的钙离子检测工具之一,在研究生物体不同组织、***的生理功能和发育机制方面做出了重要贡献;
GCaMPs是使用最广泛的GECIs之一。GCaMPs是单个绿色荧光蛋白(GFP)、钙调蛋白(CaM)和平滑肌细胞肌球蛋白轻链激酶片段M13融合的产物。GCaMPs具有一个突出的优点,它可以通过特异的启动子和定位序列实现在特定类型细胞或亚细胞结构表达,从而检测目标细胞或亚细胞结构内的钙离子信号的变化,记录生理条件下或给予外源性刺激下不同细胞的反应方式、信号的起源和传递。应用于钙离子成像的斑马鱼心脏标记品系的建立,是利用CRISPR/Cas9基因编辑技术将GCaMP5G敲入野生型斑马鱼myl7基因7号外显子上,通过体式荧光显微镜、PCR、测序鉴定斑马鱼基因型,从而建立的一种新的斑马鱼心脏标记品系。这种品系可以研究斑马鱼心脏传导***的发育,根据荧光反应的变化分析某些传导缺陷引起的心律失常的成因和机制。另外,此品系的建立对于研究其他钙离子参与的疾病、分析疾病发生发展的基础和病理生理机制提供了良好的动物模型,也为探讨药物的生理和药理学机制、评价新药的药理作用和毒性提供了有效的理论依据,在医学上具有十分重要的意义。
发明内容
本发明的目的在于提供一种应用于钙离子成像的斑马鱼心脏特异标记品系。斑马鱼与人类心脏发育过程中的基因、信号通路有高度同源性,该模型能够帮助我们研究人类心脏疾病病理生理机制和进行心脏疾病治疗药物筛选的理想模型;
本发明的另一目的在于提供上述斑马鱼品系的构建方法;
本发明所采用的技术方案,具体按照以下步骤实施:
步骤1、基于CRISPR/Cas9技术构建针对野生型斑马鱼myl7基因7号外显子的特异性sgRNA,所述sgRNA的基因序列如PrimerF所示,待敲入的编码钙离子浓度指示剂的GCaMP5G序列如图1所示;
步骤2、合成一对5’C6氨基修饰引物,5’端修饰引物为myl7基因7号外显子编码区终止密码子前的54 bp及GCaMP5G的5’端22 bp,3’端修饰引物为myl7基因7号外显子编码区终止密码子后的54 bp及GCaMP5G的3’端24 bp,引物序列如PrimerF2、PrimerR2所示;
步骤3、合成两对检测引物:F1、R1引物用以检测靶位点是否有效果,F11、R11用以检测外源DNA序列是否正确***,两边同源臂是否打靶正确;
步骤4、以质粒“pTol2-cmlc2-GCaMP5G”DNA为模板,用5’C6氨基修饰的引物进行PCR扩增,获得含有myl7同源臂的GCaMP5G序列,即GCaMP5G基因敲入序列;
步骤5、将步骤1中有活性的sgRNA、步骤4中的GCaMP5G基因敲入序列和Cas9蛋白共同注射进入斑马鱼受精卵中,获得F0代斑马鱼;
步骤6、将步骤5中F0代培养48 h,在体式荧光显微镜下筛选出阳性F0代斑马鱼;
步骤7、将步骤6中性成熟的阳性F0代斑马鱼与野生型斑马鱼交配繁殖,获得F1代杂合子斑马鱼,在体式荧光显微镜下选出可遗传的F1代斑马鱼,再通过PCR、Sanger测序鉴定基因型;
步骤8、将步骤7获得的性成熟F1代杂合子斑马鱼近交获得F2代斑马鱼,在体式荧光显微镜下镜筛选出F2代纯合子斑马鱼,即为本发明构建的一种应用于钙离子成像的斑马鱼心脏特异标记品系;
2.根据权利要求1所述的一种应用于钙离子成像的斑马鱼心脏特异标记品系的构建方法,其特征在于,所述步骤5中sgRNA的终浓度为40~60 ng/μL,打靶载体的终浓度为20~100 ng/μL,Cas9蛋白的注射浓度为700 ng/μL。
附图说明
图1为本发明使用的GCaMP5G蛋白的基因编码序列图;
图2为本发明使用的pTol2-cmlc2-GCaMP5G质粒图;
图3为本发明CRISPR/Cas9打靶***的流程图;
图4为本发明验证GCaMP5G敲入效果的测序峰图;
图5为本发明阳性F1代斑马鱼的电泳结果图;
图6为本发明阳性F1代斑马鱼5’端的序列比对图;
图7为本发明阳性F1代斑马鱼3’端的序列比对图;
图8为本发明阳性F2代斑马鱼荧光图;
图9为本发明F2代斑马鱼三引物法的电泳结果图。
具体实施方式
下面结合附图和具体的实施例,对本发明作详细描述;
实施例1:
1)分别设计CRISPR/Cas9基因敲入靶位点和检测引物
a、在National Center for Biotechnology Information(NCBI)上查询斑马鱼myl7基因的基因组和mRNA序列,根据CRISPR/Cas9基因编辑原理和myl7基因组序列的结构特点,选择myl7基因的打靶敲入位点(F序列中下划线所示);
设计合成特异性靶位点PCR引物如下:
F(靶位点正向引物):
TGTAATACGACTCACTATAGGcacatggtgatgaaaaagGTTTTAGAGCTAGAAATAGC
R(靶位点反向引物):
AAGCACCGACTCGGTGCCACT
b、在National Center for Biotechnology Information(NCBI)上查询斑马鱼myl7基因的基因组mRNA序列,根据CRISPR靶位点上下游约300 bp的基因组区域,利用Primer 5.0软件设计引物F1、R1用以检测靶位点是否有效果;
PCR检测引物:
F1:5’-CCCTTTTCACCAGACTGGCTAC-3’
R1:5’-AGACCAAGCACAGCAACTGAGT-3’
c、利用Primer 5.0软件分别在GCaMP5G序列5’端和3’端约300 bp的区域,设计引物F11、R11,结合靶位点检测引物F1、R1检测F1代阳性斑马鱼的序列***情况;
PCR检测引物:
F11:5’-CACGTGATGACAAACCTTGG-3’
R11:5’-GTTGTGGCGGATCTTGAAGT-3’
2)构建sgRNA表达载体以及特异性sgRNA体外合成
a、首先将sgRNA骨架克隆到p42250载体上;
b、特异性sgRNA体外合成
用BsaⅠ限制性内切酶线性化此质粒;酶切反应总体积为20 μL,体系如下:
混匀后于37℃水浴,酶切4 h以上;
c、以线性化的p42250载体为模板,通过下面特异性引物进行PCR扩增,获得用于特异性sgRNA合成的双链DNA;
正向特异性靶位点引物F:T7启动子19 bp + 靶序列18 bp + sgRNA上游骨架20bp;
反向引物R:21 bpsgRNA下游骨架;
50 μL高保真PCR反应体系如下:
95℃预变性5 min,95℃变性30 sec,60℃退火30 sec,72℃延伸30 sec,72℃延伸8 min,35个循环。待反应结束后,取1μL样品点样于1.2%琼脂糖凝胶,进行电泳检测,凝胶成像***拍摄电泳结果;
d、检测确定电泳条带正确之后,进行琼脂糖凝胶DNA回收,纯化回收PCR产物;
e、测定纯化的DNA浓度,再以此DNA为模板,用20 μL体系进行体外转录,合成特异性sgRNA;转录实验中所用Tip头、EP管均为RNase-Free产品,具体操作如下:
20 μL体外转录反应体系:
将反应物全部加入1.5 mL RNase-Free的EP管中,混匀之后,于37℃水浴2.5 h;然后向转录体系中加入1 μL TURBO DNase,放置于37℃水浴锅中反应30 min,用以消化DNA模板;再取1μL转录终产物,即sgRNA进行琼脂糖凝胶电泳,以检测转录效率;
f、特异性sgRNA的纯化
用RNeasy Mini kit试剂盒纯化转录成功的sgRNA,保存于-80℃;吸取纯化后的sgRNA溶液1 μL进行浓度测定;
3)设计和合成敲入序列
a、在National Center for Biotechnology Information(NCBI)上查询斑马鱼基因组myl7基因的7号外显子(即最后一个外显子)编码序列终止密码子附近的序列,根据该序列上的打靶位点分别设计5’和3’端的同源臂(引物序列中大写字母所示),并分别将同源臂与GCaMP5G基因编码序列对应的5’和3’端前20 bp(引物序列中小写字母所示)一起合成5’C6氨基修饰引物,用于PCR扩增pTol2-cmlc2-GCaMP5G载体上的GCaMP5G序列;
PCR引物如下:
F2(正向引物):5’→3’
GATTATAAGTCGCTTTGTTACATTATCACACATGGTGATGAAAAAGAaGAATCTatgggttctcatcatcatcatc
R2(反向引物):5’→3’
GTGAATGTTGAACTGAACAGATATGCAGACATAAACTGAGACATTTTCCTTTCTtcacttcgctgtcatcatttgtac
b、以pTol2-cmlc2-GCaMP5G载体为模板,通过5’C6氨基修饰引物进行PCR扩增,获得打靶载体;
正向5’C6氨基修饰引物F2:myl7基因54 bp + GCaMP5G序列22 bp;
反向5’C6氨基修饰引物R2:myl7基因54 bp + GCaMP5G序列24 bp;
50 μL高保真PCR反应体系如下:
95℃预变性5 min,95℃变性30 sec,60℃退火30 sec,72℃延伸2 min,72℃延伸8min,35个循环。待反应结束后,取1μL样品点样于1.2%琼脂糖凝胶,进行电泳检测,凝胶成像***拍摄电泳结果;
c、检测确定电泳条带正确之后,进行琼脂糖凝胶DNA回收,纯化回收PCR产物;
d、测定纯化的DNA浓度,此纯化产物即为打靶载体,将产物保存于-20℃;
4)斑马鱼胚胎的显微注射
在受精后30 min 之内,用吸管吸取胚胎转移至用琼脂糖制作的显微注射专用培养皿中,在进行显微注射之前,将sgRNA、打靶载体和Cas9蛋白配成混合液充分混匀,其中,sgRNA的终浓度为40~60 ng/μL,打靶载体的终浓度为20~100 ng/μL,Cas9蛋白的注射终浓度为700 ng/μL,注射约1.8 nL混合液于一细胞期的受精卵内;注射后的受精卵放置于E3水(5 mmol/L NaCl,0.17 mmol/L KCl,0.33 mmol/L CaCl2,0.33 mmol/L MgSO4)中,28℃培养箱孵化48 h;在体式荧光显微镜下观察胚胎,筛选带荧光的胚胎;
5)Sanger测序检测靶位点的有效性
对斑马鱼胚胎进行显微注射之后,随机挑选早期胚胎,检测myl7基因是否存在突变,可以提前确认选择的靶位点是否有效,显微注射操作是否规范;
a、提取斑马鱼基因组
斑马鱼胚胎受精50小时后(50 hpf),分别收集野生型(作对照)和注射后胚胎于1.5 mL Ep管中,每管5颗胚胎,按照下述方法提取基因组DNA,具体步骤如下:
(1)向装有胚胎的Ep管中加入100 μL细胞裂解液(0.2 mol/L NaCl,5 mmol/LEDTA,0.1 mol/L Tris-HCl,0.2%SDS)和1 μL蛋白酶K(10 mg/mL),放置于65℃恒温箱中裂解过夜;
(2)裂解完成后,放在振荡器上充分震荡,加入等体积预冷的异丙醇,充分颠倒混匀,于4℃条件下,12000 rpm离心10 min,弃掉上清;
(3)加入75%乙醇200 μL,于4℃条件下,12000 rpm离心5 min,弃掉上清,室温干燥8~10min;
(4)加入10 μL去离子水,充分混匀,即得斑马鱼基因组DNA;
b、PCR扩增
提取基因组DNA后,使用PCR检测引物F1、R1进行扩增;
20 μL常规PCR反应体系如下:
94℃预变性5 min,94℃变性30 sec,60℃退火30 sec,72℃延伸30 sec,72℃延伸8 min,30个循环。待反应结束后,取3μL样品点样于1.2%琼脂糖凝胶,进行电泳检测,凝胶成像***拍摄电泳结果;
c、将剩余PCR产物进行Sanger测序,由测序的峰图来初步获得***或缺失的信息;
6)筛选可遗传的杂合子斑马鱼F1代
a、将性成熟的阳性F0代斑马鱼分别与野生型斑马鱼杂交得到F1代胚胎,置于28℃培养48 h,在体式荧光显微镜下筛选可遗传的阳性F1代斑马鱼,此阳性F1代为杂合子;
b、将阳性F1代剪尾,单独提取基因组,使用PCR检测引物F1、R11进行扩增,得到DNA片段a,使用PCR检测引物F11、R1进行扩增,得到DNA片段b;
20 μL常规PCR反应体系如下:
94℃预变性5 min,94℃变性30 sec,60℃退火30 sec,72℃延伸30 sec,72℃延伸8 min,30个循环。待反应结束后,取3μL样品点样于1.2%琼脂糖凝胶,进行电泳检测,凝胶成像***拍摄电泳结果;
c、阳性F1代DNA片段a大小为441bp,DNA片段b大小为470bp。电泳图显示DNA片段b与预计大小不一致,因此将剩余PCR产物进行Sanger测序,由序列比对来检测验证外源DNA序列是否正确***,两边同源臂是否打靶正确;
7)获得可遗传的纯合子斑马鱼F2代
a、将性成熟的阳性F1代斑马鱼雌雄近交得到F2代胚胎,置于28℃培养48 h,在体式荧光显微镜下筛选阳性F2代斑马鱼;
b、由于筛选到的阳性F2代斑马鱼可能是杂合子也可能是纯合子,可以通过对每条阳性F2代斑马鱼成鱼进行剪尾鉴定,筛选出纯合子阳性F2代斑马鱼;
c、使用三引物法鉴定F2代斑马鱼。首先将F2代斑马鱼剪尾,单独提取基因组,然后使用PCR检测引物F1、R1、R11进行扩增;
20 μL常规PCR反应体系如下:
94℃预变性5 min,94℃变性30 sec,60℃退火30 sec,72℃延伸30 sec,72℃延伸8 min,30个循环。待反应结束后,取3μL样品点样于1.2%琼脂糖凝胶,进行电泳检测,凝胶成像***拍摄电泳结果;
d、根据凝胶成像图鉴定F2代斑马鱼的基因型
根据三引物法可以判断:野生型只扩增出539 bp条带,杂合子扩增539 bp和441bp两条条带,纯合子只扩增441 bp条带;
图1为GCaMP5G蛋白的基因编码序列图;图2为pTol2-cmlc2-GCaMP5G质粒图;图3为CRISPR/Cas9打靶***的流程图;图4为验证GCaMP5G敲入效果的测序峰图,靶位点序列下游可以观察到双峰,说明靶位点有效果;图5为阳性F1代斑马鱼的电泳结果图,阳性F1代DNA片段a大小为441bp,DNA片段b大小为539 bp;图6为本发明阳性F1代斑马鱼5’端的序列比对图,5’端比对结果显示靶位点下游含有GCaMP5G序列,与预计的敲入序列一致;图7为本发明阳性F1代斑马鱼3’端的序列比对图,3’端比对结果显示敲入额外的66 bp,此序列包含3’端同源臂;图8为阳性F2代斑马鱼荧光图,心脏部位尤其是心室有明显的荧光;图9为本发明F2代斑马鱼三引物法的电泳结果图,野生型(1、2)只有一条539 bp的电泳条带,杂合子(3、4)有一条539 bp和一条441 bp的电泳条带,纯合子(5)只有一条441 bp的电泳条带;
以上所述,仅是本发明的较佳实施例,并非对本发明做任何形式上的限制,凡是依据本发明的技术实质上对以上实施例所作的任何简单修改、等同变化,均落入本发明的保护范围之内。
序列表
<110> 湖南师范大学
<120> 一种应用于钙离子成像的斑马鱼心脏特异标记品系的建立
<160> 17
<170> SIPOSequenceListing 1.0
<210> 18
<211> 18
<212> DNA
<213> Danio rerio
<400> 18
cacatggtga tgaaaaag 18
<210> 1
<211> 59
<212> DNA
<213> p42250载体(Escherichia coli)
<400> 1
tgtaatacga ctcactatag gcacatggtg atgaaaaagg ttttagagct agaaatagc 59
<210> 2
<211> 21
<212> DNA
<213> p42250载体(Escherichia coli)
<400> 2
aagcaccgac tcggtgccac t 21
<210> 3
<211> 22
<212> DNA
<213> 斑马鱼(Danio rerio)
<400> 3
cccttttcac cagactggct ac 22
<210> 4
<211> 22
<212> DNA
<213> 斑马鱼(Danio rerio)
<400> 4
agaccaagca cagcaactga gt 22
<210> 5
<211> 20
<212> DNA
<213> pTol2-cmlc2-GCaMP5G(Escherichia coli)
<400> 5
cacgtgatga caaaccttgg 20
<210> 6
<211> 20
<212> DNA
<213> pTol2-cmlc2-GCaMP5G(Escherichia coli)
<400> 6
gttgtggcgg atcttgaagt 20
<210> 10
<211> 54
<212> DNA
<213> 斑马鱼(Danio rerio)
<400> 10
gattataagt cgctttgtta cattatcaca catggtgatg aaaaagaaga atct 54
<210> 11
<211> 54
<212> DNA
<213> 斑马鱼(Danio rerio)
<400> 11
gtgaatgttg aactgaacag atatgcagac ataaactgag acattttcct ttct 54
<210> 7
<211> 76
<212> DNA
<213> 斑马鱼(Danio rerio)
<400> 7
gattataagt cgctttgtta cattatcaca catggtgatg aaaaagaaga atctatgggt 60
tctcatcatc atcatc 76
<210> 8
<211> 78
<212> DNA
<213> 斑马鱼(Danio rerio)
<400> 8
gtgaatgttg aactgaacag atatgcagac ataaactgag acattttcct ttcttcactt 60
cgctgtcatc atttgtac 78
<210> 9
<211> 1353
<212> DNA
<213> pTol2-cmlc2-GCaMP5G(Escherichia coli)
<400> 9
atgggttctc atcatcatca tcatcatggt atggctagca tgactggtgg acagcaaatg 60
ggtcgggatc tgtacgacga tgacgataag gatctcgcca ccatggtcga ctcatcacgt 120
cgtaagtgga ataagacagg tcacgcagtc agagctatag gtcggctgag ctcactcgag 180
aacgtctata tcaaggccga caagcagaag aacggcatca aggcgaactt caagatccgc 240
cacaacatcg aggacggcgg cgtgcagctc gcctaccact accagcagaa cacccccatc 300
ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcgtgcagtc caaactttcg 360
aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg 420
atcactctcg gcatggacga gctgtacaag ggcggtaccg gagggagcat ggtgagcaag 480
ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 540
ggccacaagt tcagcgtgtc cggcgagggt gagggcgatg ccacctacgg caagctgacc 600
ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 660
ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 720
ttcaagtccg ccatgcccga aggctacatc caggagcgca ccatcttctt caaggacgac 780
ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 840
gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 900
aacctgccgg accaactgac tgaagagcag atcgcagaat ttaaagaggc tttctcccta 960
tttgacaagg acggggatgg gacaataaca accaaggagc tggggacggt gatgcggtct 1020
ctggggcaga accccacaga agcagagctg caggacatga tcaatgaagt agatgccgac 1080
ggtgacggca caatcgactt ccctgagttc ctgacaatga tggcaagaaa aatgaaatac 1140
acagacagtg aagaagaaat tagagaagcg ttccgtgtgt ttgataagga tggcaatggc 1200
tacatcagtg cagcagagct tcgccacgtg atgacaaacc ttggagagaa gttaacagat 1260
gaagaggttg atgaaatgat cagggaagca gacatcgatg gggatggtca ggtaaactac 1320
gaagagtttg tacaaatgat gacagcgaag tga 1353
<210> 15
<211> 441
<212> DNA
<213> 斑马鱼(Danio rerio)
<400> 15
cccttttcac cagactggct acgggcctgt catatcatca gctataagac tgaatctttg 60
gtgatgttct gatcttgatc gtatgtgttt caggttgacc aggcttttgc agtggctcca 120
atagacgtgg ctggaaatat tgattataag tcgctttgtt acattatcac acatggtgat 180
gaaaaagaag aatctatggg ttctcatcat catcatcatc atggtatggc tagcatgact 240
ggtggacagc aaatgggtcg ggatctgtac gacgatgacg ataaggatct cgccaccatg 300
gtcgactcat cacgtcgtaa gtggaataag acaggtcacg cagtcagagc tataggtcgg 360
ctgagctcac tcgagaacgt ctatatcaag gccgacaagc agaagaacgg catcaaggcg 420
aacttcaaga tccgccacaa c 441
<210> 14
<211> 470
<212> DNA
<213> 斑马鱼(Danio rerio)
<400> 14
cacgtgatga caaaccttgg agagaagtta acagatgaag aggttgatga aatgatcagg 60
gaagcagaca tcgatgggga tggtcaggta aactacgaag agtttgtaca aatgatgaca 120
gcgaagtgaa gaaaggaaaa tgtctcagtt tatgtctgca tatctgttca gttcaacatt 180
cacatcagca gcttttaata ttaaaacatt tgtgttgcat ttcaacacaa tactaatgtc 240
tgttttatat acaaaataaa cattagatta cttgactgag cttaagagaa gaccatgaga 300
agagataatt gtcatcttat ttattctgtc tgtaactgct gccccctaca gactgttaat 360
tgcaattaat aactagacat ttggtttaag gcccttaaat tccttgacta tgagtagagc 420
atcatctcaa acctataata aacttcatac tcagttgctg tgcttggtct 470
<210> 16
<211> 539
<212> DNA
<213> 斑马鱼(Danio rerio)
<400> 16
cccttttcac cagactggct acgggcctgt catatcatca gctataagac tgaatctttg 60
gtgatgttct gatcttgatc gtatgtgttt caggttgacc aggcttttgc agtggctcca 120
atagacgtgg ctggaaatat tgattataag tcgctttgtt acattatcac acatggtgat 180
gaaaaagagg aatcttgaag aaaggaaaat gtctcagttt atgtctgcat atctgttcag 240
ttcaacattc acatcagcag cttttaatat taaaacattt gtgttgcatt tcaacacaat 300
actaatgtct gttttatata caaaataaac attagattac ttgactgagc ttaagagaag 360
accatgagaa gagataattg tcatcttatt tattctgtct gtaactgctg ccccctacag 420
actgttaatt gcaattaata actagacatt tggtttaagg cccttaaatt ccttgactat 480
gagtagagca tcatctcaaa cctataataa acttcatact cagttgctgt gcttggtct 539
<210> 15
<211> 417
<212> DNA
<213> 斑马鱼(Danio rerio)
<400> 15
gggaagttca tctcagctat aagactgaat ctttggtgat gttctgatct tgatcgtatg 60
tgtttcaggt tgaccaggct tttgcagtgg ctccaataga cgtggctgga aatattgatt 120
ataagtcgct ttgttacatt atcacacatg gtgatgaaaa agaagaatct atgggttctc 180
atcatcatca tcatcatggt atggctagca tgactggtgg acagcaaatg ggtcgggatc 240
tgtacgacga tgacgataag gatctcgcca ccatggtcga ctcatcacgt cgtaagtgga 300
ataagacagg tcacgcagtc agagctatag gtcggctgag ctcactcgag aacgtctata 360
tcaaggccga caagcagaag aacggcatca aggcgaactt caagatccgc cacaaca 417
<210> 16
<211> 509
<212> DNA
<213> 斑马鱼(Danio rerio)
<400> 16
cgggttcgga tgatgaggtt gatgaatgat cagggaagca gacatcgatg gggatggtca 60
ggtaaactac gaagagtttg tacaaatgat gacagcgaag tgaagaaagg aaaatgtctc 120
agtttatgtc tgcatatctg ttcagttcaa cattcaagag gaatcttgaa gaaaggaaaa 180
tgtctcagtt tatgtctgca tatctgttca gttcaacatt cacatcagca gcttttaata 240
ttaaaacatt tgtgttgcat ttcaacacaa tactaatgtc tgttttatat acaaaataaa 300
cattagatta cttgactgag cttaagagaa gaccatgaga agagataatt gtcatcttat 360
ttattctgtc tgtaactgct gccccctaca gactgttaat tgcaattaat aactagacat 420
ttggtttaag gcccttaaat tccttgacta tgagtagagc atcatctcaa acctataata 480
aacttcatac tcagttgctg tgcttggtc 509
Claims (2)
1.一种应用于钙离子成像的斑马鱼心脏特异标记品系的建立,其特征在于,按照以下步骤进行实施:
步骤1、基于CRISPR/Cas9技术构建针对野生型斑马鱼myl7基因7号外显子的特异性sgRNA,所述sgRNA的基因序列如PrimerF所示,待敲入的编码钙离子浓度指示剂的GCaMP5G序列如图1所示;
步骤2、合成一对5’C6氨基修饰引物,5’端修饰引物为myl7基因7号外显子编码区终止密码子前的54 bp及GCaMP5G的5’端22 bp,3’端修饰引物为myl7基因7号外显子编码区终止密码子后的54 bp及GCaMP5G的3’端24 bp,引物序列如PrimerF2、PrimerR2所示;
步骤3、合成两对检测引物:F1、R1引物用以检测靶位点是否有效果,F11、R11用以检测外源DNA序列是否正确***,两边同源臂是否打靶正确;
步骤4、以质粒“pTol2-cmlc2-GCaMP5G”DNA为模板,用5’C6氨基修饰的引物进行PCR扩增,获得含有myl7同源臂的GCaMP5G序列,即GCaMP5G基因敲入序列;
步骤5、将步骤1中有活性的sgRNA、步骤4中的GCaMP5G基因敲入序列和Cas9蛋白共同注射进入斑马鱼受精卵中,获得F0代斑马鱼;
步骤6、将步骤5中F0代培养48 h,在体式荧光显微镜下筛选出阳性F0代斑马鱼;
步骤7、将步骤6中性成熟的阳性F0代斑马鱼与野生型斑马鱼交配繁殖,获得F1代杂合子斑马鱼,在体式荧光显微镜下选出可遗传的F1代斑马鱼,再通过PCR、Sanger测序鉴定基因型;
步骤8、将步骤7获得的性成熟F1代杂合子斑马鱼近交获得F2代斑马鱼,在体式荧光显微镜下镜筛选出F2代纯合子斑马鱼,即为本发明构建的一种应用于钙离子成像的斑马鱼心脏特异标记品系。
2.根据权利要求1所述的一种应用于钙离子成像的斑马鱼心脏特异标记品系的构建方法,其特征在于,所述步骤5中sgRNA的终浓度为40~60 ng/μL,打靶载体的终浓度为20~100 ng/μL,Cas9蛋白的注射终浓度为700 ng/μL。
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CN114540418A (zh) * | 2022-02-28 | 2022-05-27 | 福建省妇幼保健院 | 斑马鱼心肌特异表达线粒体质量指示探针品系的构建 |
CN114868707A (zh) * | 2022-06-02 | 2022-08-09 | 浙江大学 | 一种代谢性脑病和心律失常疾病的斑马鱼模型及其应用 |
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CN114480497A (zh) * | 2022-02-28 | 2022-05-13 | 湖南师范大学 | 一种ep400基因敲除斑马鱼心力衰竭模型的构建及其应用的方法 |
CN114540418A (zh) * | 2022-02-28 | 2022-05-27 | 福建省妇幼保健院 | 斑马鱼心肌特异表达线粒体质量指示探针品系的构建 |
CN114480497B (zh) * | 2022-02-28 | 2023-09-15 | 湖南师范大学 | 一种ep400基因敲除斑马鱼心力衰竭模型的构建及其应用的方法 |
CN114868707A (zh) * | 2022-06-02 | 2022-08-09 | 浙江大学 | 一种代谢性脑病和心律失常疾病的斑马鱼模型及其应用 |
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