CN111118009A - 敲除斑马鱼p2rx2基因的方法 - Google Patents
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Abstract
本发明涉及基因敲除技术领域,尤其涉及敲除斑马鱼p2rx2基因的方法。本发明通过CRISPR/Cas9基因编辑技术,在斑马鱼p2rx2基因上设计合适的打靶位点,在体外合成特异性sgRNA和Cas9‑mRNA用于斑马鱼的基因编辑。本发明能更高效且更精确地沉默特定基因,且制作简单、成本低,且可同时对靶基因上多个位点进行剪切,沉默任意数目的单个基因,本发明还提供了基因敲除选育p2rx2基因缺失型斑马鱼的方法,该方法构得的斑马鱼模型,有助于进一步揭示耳泡以及毛细胞形态发生的整个过程以及调控这些过程的分子机制,在医学上先天性耳聋致病机理的理解和基因矫正的治疗方案的研发中具有十分重要的意义。
Description
技术领域
本发明涉及基因敲除技术领域,尤其涉及敲除斑马鱼p2rx2基因的方法。
背景技术
p2rx2基因位于斑马鱼第5号染色体上,包含12个外显子和11个内含子,cDNA全长1292bp,编码400氨基酸,p2rx2包含有4个进化上保守的功能结构域,同时通过基因差异表达谱分析和基因组关联分析等,发现p2rx2在人类胚胎早期的多个组织中都有表达,特别是耳泡中强烈表达。P2RX2基因突变可引起非综合征型常染色体显性遗传性耳聋(DFNA41),P2RX2基因为噪声易感基因的候选基因,患者对噪声高度敏感。
斑马鱼与人类耳道发育过程中的基因、信号通路有高度同源性,且p2rx2基因进化上较为保守,研究发现p2rx2在斑马鱼胚胎早期表达量特别高。而且,与其他动物模型相比,斑马鱼具有个体小、易于饲养、发育快、繁殖能力强、体外受精、胚胎体外发育且透明等优点。因此,通过干扰掉斑马鱼体内的p2rx2基因,并且通过遗传学手段研究其功能,有助于进一步揭示耳道形态发生的整个过程以及调控这些过程的分子机制,在医学上耳道疾病病理的理解和新的治疗方案的研发中具有十分重要的意义。且干扰掉p2rx2基因,并且通过遗传学手段研究其功能,有助于进一步揭示耳泡以及毛细胞形态发生的整个过程以及调控这些过程的分子机制,在医学上先天性耳聋致病机理的理解和基因矫正的治疗方案的研发中具有十分重要的意义。
基因打靶技术起源于20世纪80年代末,是一种通过对基因组进行定点修饰来研究基因功能的重要方法手段,也可用于治疗人类的各种遗传性疾病。该技术主要是利用缺失突变、基因灭活、染色体大片段删除以及外源基因导入等方式来改变生物的遗传信息,并且在生殖系中稳定遗传后表达突变性状,从而研究生物体内特定基因在生长发育过程中的作用,所以这类技术手段已成为现代分子生物学研究热点。传统的基因打靶技术是建立在胚胎干细胞(ESC)和同源重组技术的基础之上,故打靶技术效率极低。2013年初,一种全新的人工核酸内切酶clustered regularly interspaced shortpalindromic repeats(CRISPR)/CRISPR-associated(Cas)9,能更高效且更精确地在生物体基因组中沉默特定基因,且制作简单、成本低,且可同时对靶基因上多个位点进行剪切,沉默任意数目的单个基因。
但是目前通用的检测CRISPR/Cas9突变方法应用于斑马鱼的基因敲除存在一定的缺陷,例如:在F0代对斑马鱼进行剪尾,并且做TA克隆,Sanger测序,需要花费大量的时间、试剂以及测序成本,并且F0代测序得到的不一定能够遗传到下一代。并且,CRISPR/Cas9技术所需的步骤较多,成本太高,脱靶率相对也较高。
发明内容
有鉴于此,本发明要解决的技术问题在于提供敲除斑马鱼p2rx2基因的方法,该方法采用cloning free策略进行斑马鱼p2rx2基因的敲除,脱靶率较低。
本发明提供了靶向斑马鱼p2rx2基因的gRNA,包括启动子元件、靶序列和骨架序列;其中,靶序列如SEQ ID NO:1或SEQ ID NO:2所示。
本发明所述启动子元件的序列如SEQ ID NO:3所示;所述骨架序列如SEQ ID NO:4所示。
在一些具体实施例中,所述靶向斑马鱼p2rx2基因的gRNA包括SEQ ID NO:5~6所示的两条gRNA。
本发明所述的靶序列针对斑马鱼p2rx2基因的3号外显子进行设计,本发明共设计了两个编辑的靶位点,分别为靶位点a和靶位点b。靶位点a(利用T7启动子进行转录)的选择遵循以下标准:5’-(N)20-NGG-3’,保证靶位点的3’端是NGG;靶位点b选择遵循以下标准:5’-GG-(N)18-NGG-3’其中5’端的GG二核苷酸是T7启动子的一部分,保证靶位点的3’端是NGG;靶点的选择位置在p2rx2基因的茎环区域内。本发明所设计的靶向斑马鱼p2rx2基因的gRNA具有更好的特异性,相对于其它gRNA而言,采用本发明设计的gRNA脱靶率较低,配合cloning free CRISPR/Cas9获得了良好的斑马鱼p2rx2基因的敲除效果。
本发明中,所述靶向斑马鱼p2rx2基因的gRNA的制备方法包括:
步骤1:以合成的DNA为模板,分别以两对特异性引物进行扩增,获得双链DNA;
步骤2:将所述双链DNA进行转录后,纯化过得gRNA。
其中,合成的DNA的序列如SEQ ID NO:10所示。
扩增获得SEQ ID NO:5所示gRNA的特异性引物中,上游引物如SEQ ID NO:11所示;下游引物如SEQ ID NO:13所示;
扩增获得SEQ ID NO:6所示gRNA的特异性引物中,上游引物如SEQ ID NO:12所示;下游引物如SEQ ID NO:13所示。
所述转录的体系包括:
本发明还提供了靶向斑马鱼p2rx2基因的敲除试剂,其包括本发明所述的gRNA和Cas9 mRNA。
本发明中,所述Cas9 mRNA针对斑马鱼进行密码子优化,序列如SEQ ID NO:9。
本发明中,所述敲除试剂中包括Nuclease free H2O和如下浓度的:
MgCl2 10mmol/L;
Cas9 mRNA 150ng/μL;
gRNA 各20ng/μL。
本发明所述的敲除试剂在构建p2rx2基因缺失型斑马鱼模型中的应用。
本发明还提供了一种构建p2rx2基因缺失型斑马鱼模型的方法,其将所述的敲除试剂接入一细胞期的斑马鱼受精卵内,孵化获得p2rx2基因缺失型斑马鱼模型。
本发明中,每个受精卵给予1.5~2.0nL所述的敲除试剂。一些实施例中,每个受精卵给予1.8nL所述的敲除试剂
所述孵育后还包括筛选的步骤;所述筛选包括:对受精36h后的胚胎进行筛选,或对成鱼进行筛选;
所述筛选包括对基因组DNA进行扩增后进行Sanger测序或根据电泳结果进行筛选;扩增产物包含227bp和322bp大小的条带则p2rx2基因缺失,扩增产物仅有322bp大小的片段则p2rx2基因未缺失;所述扩增的引物序列如SEQ ID NO:7~8所示。
所述孵化获得的p2rx2基因缺失斑马鱼为F0代;所述方法还包括传代的步骤;
F1代斑马鱼由F0代斑马鱼与野生型的斑马鱼杂交获得;
F2代斑马鱼由F1代突变体杂交获得。
本发明通过CRISPR/Cas9基因编辑技术,在斑马鱼的p2rx2基因上设计合适的打靶位点,在体外合成的特异性sgRNA(终浓度20ng/μL)和Cas9-mRNA(终浓度150ng/μL),显微共注射进入斑马鱼一细胞内,胚胎培养36h后,通过选取胚胎进行基因型分析,鉴定所设打靶位点的有效性。本发明能更高效且更精确地在生物体基因组中沉默特定基因,且制作简单、成本低,且可同时对靶基因上多个位点进行剪切,沉默任意数目的单个基因,且干扰掉p2rx2基因,并且通过遗传学手段研究其功能,有助于进一步揭示耳形态发生的整个过程以及调控这些过程的分子机制,在医学上对耳聋疾病病理的理解和新的治疗方案的研发中具有十分重要的意义。
附图说明
图1为CRISPR/Cas9打靶***的原理图;
图2为p2rx2基因上靶位点的结构图;
图3为斑马鱼F0代T7E1酶切电泳结果图;
图4为斑马鱼F1代电泳结果图;
图5为缺失型和WT型基因序列正向对比;
图6为靶位点附近缺失对比。
具体实施方式
本发明提供了敲除斑马鱼p2rx2基因的方法,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。
本发明采用的试材皆为普通市售品,皆可于市场购得。
下面结合实施例,进一步阐述本发明:
实施例1:
1)设计CRISPR/Cas9基因敲除靶位点和检测引物
在National Center for Biotechnology Information(NCBI)上查询斑马鱼p2rx2基因的基因组DNA序列,在网站SMART(http://smart.embl-heidelberg.de/)上分析其功能结构域,根据CRISPR/Cas敲除原理,在网站The ZiFiT Targeter(http://zifit.partners.org/ZiFiT/)上设计p2rx2基因的靶位点。靶点的选择必须遵循此标准:5’-GG-(N)18-NGG-3’。其中5’端的GG二核苷酸是T7启动子的一部分,设计靶位点时可以不受此限制,但是必须保证靶位点的3’端是NGG。靶点的选择位置必须在基因的结构域内,以确保靶位点碱基的***或者缺失可以影响p2rx2基因的整个结构域,从而来改变基因的表达。
两对特异性靶位点PCR引物如下:
F1(靶位点a正向引物):
GCGTAATACGACTCACTATAGGTGGGGTTTTGCTGTGTGCGTTTTAGAGCTAGAAATAG(SEQ IDNO:11)
F2(靶位点b正向引物):
GCGTAATACGACTCACTATAGGAATACGTCAGACCATCTGGTTTTAGAGCTAGAAATAG(SEQ IDNO:12)
R(共用的反向引物):AAGCACCGACTCGGTGCCACT(SEQ ID NO:13)
PCR检测引物
PCR检测引物上下游引物分别位于p2rx2第一号和第二号内含子上:
F(5’-TTGTTTGGCATGTATCTGGG-3’)(SEQ ID NO:7)
R(5’-CCACTGTCACCCCATATAGTA-3(SEQ ID NO:8)
通用模板序列
TTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT(SEQ ID NO:10)
2)gRNA体外合成
a,以合成的DNA为模板,通过下面特异性引物进行PCR,扩增出用于特异性gRNA合成的双链DNA。
正向特异性靶位点引物F1或F2:T7启动子20pb靶序列20bp gRNA上游骨架;反向引物R:20bp gRNA下游骨架
PCR反应体系(25μL)如下:
震荡混匀之后,4℃离心,于PCR仪上进行扩增反应。反应条件为:预变性95℃3min,(变性95℃15s,退火60℃15s,延伸72℃15s)32个循环,再72℃5min。待反应结束后,离心PCR产物,取1μL样品点样于1.5%琼脂糖凝胶上进行电泳,凝胶成像***拍摄结果。
b,检测确定条带正确之后,进行琼脂糖凝胶DNA回收,纯化回收PCR产物。
c,测定纯化的DNA浓度(尽量达到1μg),再以此DNA为模板,用20μL体系进行体外转录,合成特异性gRNA。转录实验中所用Tip头,EP管均为DEPC处理过的RNase-Free产品,具体操作如下:
体外转录反应体系(20μL):
将反应物都加入0.2mL RNase-Free的EP管中,混匀之后,于37℃水浴2h;
水浴结束后,向转录体系中加入1μLDNA酶,放置于37℃水浴锅中反应30min,以消化DNA模板,然后取1μL样品,用配制好的1.5%的琼脂糖凝胶进行电泳,以检测转录结果,若转录产物大小与预期的相符,则说明转录成功;
d,特异性gRNA的纯化
用RNeasy Mini kit试剂盒纯化转录成功的gRNA,保存于-20℃。吸取纯化后的gRNA溶液1μL进行琼脂糖凝胶电泳,以检验纯化产物,并测定纯化之后的gRNA浓度。
3)斑马鱼胚胎的显微注射
在受精后30min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Cas9 mRNA和gRNA配成混合液,充分混匀,使Cas9 mRNA的终浓度为150ng/μL,gRNA的终浓度为20ng/μL。注射约1.0nL Cas9 mRNA和gRNA混合液于一细胞期的受精卵内。注射过的受精卵放置于E3水(5mmol/L NaCl,0.33mmol/L CaCl2,0.33mmol/L MgSO4,0.17mmol/L KCl,)中,28℃孵化。在体式显微镜下观察胚胎表型,筛选正常发育的胚胎用于靶位点突变分析。
显微注射体系如下:
4)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5~10个早期胚胎,检测其p2rx2基因是否存在突变,可以提前确认此次选择的靶位点是否有效果,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36小时后(36hpf),分别收集野生型(做对照)和注射后胚胎于1.5mLEp管中(每管2颗胚胎),按照下述方法提取基因组DNA,具体步骤如下:
向装有胚胎的Ep管中加入200μL细胞裂解液,2μL蛋白酶K,放置于55℃水浴锅中裂解过夜。
裂解完成后,放在振荡器上充分震荡,加入等体积(200μL)异丙醇(预先冷却)于Ep管中,充分颠倒混匀,于4℃条件下,12000×g离心10min,倒掉上清液;
加入75%乙醇500μL,于4℃条件下,12000×g离心5min,弃上清液,室温风干20min;
加入60μL去离子水,充分吹打混匀,琼脂糖凝胶电泳检测提取效率
b、PCR扩增目的序列
提取基因组DNA之后,根据CRISPR靶位点上下游约150-200bp的基因组区域,利用Primer Premier 5.0软件设计引物序列以扩增出目的DNA片段。
PCR反应体系(30μL)如下:
震荡混匀之后,4℃离心,于PCR仪上进行扩增反应。反应条件为:预变性95℃5min,(变性95℃30s,退火60℃30s,延伸72℃30s)30个循环,再72℃8min。待反应结束后,离心PCR产物,取5μL样品点样于1.5%琼脂糖凝胶上进行电泳,检测是否有大片段缺失。
c、若无大片段缺失,则进行T7E1酶切,用1.5%琼脂糖凝胶电泳鉴定是否有小片段缺失。
d、PCR大量扩增目的序列
PCR反应体系如下:
震荡混匀之后,4℃离心,于PCR仪上进行扩增反应,反应条件为:预变性95℃5min,(变性95℃30s,退火60℃30s,延伸72℃30s)30个循环,再72℃8min。待反应结束后,离心PCR产物,取5μL样品点样于1.5%琼脂糖凝胶上进行电泳,检测PCR产物大小是否正确。
e.若PCR产物大小正确,1.5%琼脂糖凝胶电泳分离PCR产物,切下目的条带进行DNA纯化。
f.送部分纯化之后的目的DNA片段进行Sanger测序,由测序的峰图来初步获得***或缺失的信息。
4)注射两个月之后,进行剪尾鉴定,同上鉴定步骤。
5)目的序列的TA克隆
PCR初步鉴定有突变可能的目的序列再进行Sanger测序。若测序峰图有双峰,并且测序结果显示有***或缺失现象的目的序列,接下来进行TA克隆之后挑取单克隆作进一步检测。
6)质粒的Sanger测序
将双酶切检测结果显示条带大小符合预期结果的质粒送往测序,根据测序之后给出的峰图和序列,在NCBI上与标准目的序列进行对比,根据比对结果,分析出每个单克隆的突变类型。
检测结果表明36条F0代斑马鱼中,突变的有7条。
7)获得可遗传的斑马鱼突变体的F1代
通过前面一系列筛选确定了斑马鱼突变体F0代,紧接着将F0代突变体分别与野生型斑马鱼杂交得到F1代胚胎,置于28℃培养,在初期观察F1代的存活率。受精两天后,每个突变体F1代分别取10个胚胎进行突变遗传性鉴定。将每个胚胎单独提取基因组,然后PCR扩增出322bp的靶位点附近区域,观察PCR扩增是否会出现小带,PCR会出现一条227bp左右的小带,如果此突变是否可以遗传到F1代,则PCR扩增是否会出现小于227bp的小带。
如果从F1代胚胎中检测到存在突变,则将斑马鱼突变体的F1代养大至2-3个月。再分别对每条F1代斑马鱼成鱼进行剪尾,筛选F1代突变体(具体方法如前面所述)。经鉴定,26条F1代斑马鱼中有遗传效应的有3条。
8)获得斑马鱼突变体的F2代纯合子
从F1代突变体中挑选相同突变的雌鱼和雄鱼,杂交得到F2代,放置于28℃培养,受精四天后取部分胚胎进行鉴定。将每个胚胎单独提取基因组,PCR扩增出227bp靶位点附近区域,通过PCR扩增分析并测序,初步检验是否可以得到p2rx2突变体纯合子。如检验结果证明存在纯合子,则养大后再单条剪尾鉴定。经鉴定,34条F2代斑马鱼中纯合子为8条。
9)、同上可进行该基因缺失型斑马鱼的F3代纯系遗传,得到这种新的斑马鱼品系。
经传代至F3代,已经可获得稳定的纯系斑马鱼品系。附图3为斑马鱼F0代T7E1酶切电泳结果图,将F0代的成鱼进行基因型分析,T7E1酶切结果显示,E1与C1泳道相比,除322bp的目的条带外,还有酶切的带,同样,E2,E4有酶切的带,因此,1,2,4号鱼有小片段的基因缺失。
附图4为斑马鱼F1代电泳结果图,将F1代的成鱼进行基因型分析,PCR扩增结果显示,2号泳道与野生型相比,除322bp的目的条带外,还有一条227bp左右的条带,将此条带进行切胶回收,TA克隆,并测序。如图4和图5显示,将测序结果与野生型序列(322bp)进行对比,发现p2rx2靶位点b处(下划线)有碱基缺失,靶位点b处有91个碱基的缺失(缺失297个氨基酸并造成碱基移码)。由于筛选到的2号突变体的F1代的p2rx2基因部分碱基缺失造成整个基因的移码突变,改变了斑马鱼p2rx2基因的表达。从而影响斑马鱼耳泡的发育。
以上仅是本发明的优选实施方式,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。
序列表
<110> 中南大学湘雅二医院
<120> 敲除斑马鱼p2rx2基因的方法
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ccauccagua uaagaaacaa uccaaagauu uacaauuacu ggaaguauuu gauggaaaaa 2760
gaguucauca gcaaagaaaa auacaauaga uuaauaagaa auacaccacu aacaaaugaa 2820
gaacuuggcg gauucaucaa cagacaacuu guagaaacaa gacaaucaac aaaagcaauc 2880
aaagaauuau uugaaaaguu cuaccaaaaa ucaaaaauaa uaccuguaaa agcaagucuu 2940
gcaagugauu ugagaaaaga caugaauacc cuuaaaucca gagaaguaaa ugaccuucac 3000
caugcucacg augcguuuuu gaauauugua gcaggagaug uguggaaucg agaguucaca 3060
ucaaauccaa uaaauuaugu caaagaaaac agagaaggug acaagguaaa auauucguua 3120
agcaaagauu uuacaagacc ucguaaaucc aaaggaaaag uuaucuggac accugaaaaa 3180
gguagaaaau ugauuguaga uacauugaau aaaccaucag uucuaaucag caaugaaagu 3240
cauguaaaaa aaggagaguu auucaacgcu accauugcag ggaaaaagga uuacaagaaa 3300
gguaaaauau aucuuccacu aaaaaaagac gauagauuac aagauguauc gaaauaugga 3360
ggauauaagg cuauaaaugg agcguucuuu uucuugguag agcauacuaa aagcaagaaa 3420
agaauaagaa gcauagaauu auuuccguua cauuugcuua guaaauuuua ugaagauaaa 3480
aauacaguau uagauuaugc gauaaaugua uugcaauuac aagauccaaa gauaauaaua 3540
gacaaaauua auuaucguac agaaauaauu auagauaauu uuaguuauuu aauauccacu 3600
aaaucgaaug augguaguau aacuguuaaa ccaaaugagc aaauguauug gagaguugau 3660
gaaauuucga auuugaaaaa aauagaaaau aaauacaaaa aagaugccau auuaacagaa 3720
gaggauagaa aaauuaugga gaguuauauu gauaaaaucu aucaacaauu caaggcagga 3780
aaauacaaga auagacgcac uacugauaca auaauagaaa aauaugaaau aaucgaucua 3840
gacacucuag auaauaaaca auuauaccaa uuacugguag cuuuuauuuc acuuucauau 3900
aaaacaucaa auaaugcagu ggacuuuacu guaauuggac uagguacuga auguggaaag 3960
ccaagaauua cgaauuuacc ugacaacaca uaucuaguau auaaaucaau aacaggaaua 4020
uaugaaaaga ggauaagaau aaaauaa 4047
<210> 10
<211> 78
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ttttagagct agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg 60
gcaccgagtc ggtgcttt 78
<210> 11
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gcgtaatacg actcactata ggtggggttt tgctgtgtgc gttttagagc tagaaatag 59
<210> 12
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
gcgtaatacg actcactata ggaatacgtc agaccatctg gttttagagc tagaaatag 59
<210> 13
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
aagcaccgac tcggtgccac t 21
Claims (10)
1.靶向斑马鱼p2rx2基因的gRNA,包括启动子元件、靶序列和骨架序列;其中,靶序列如SEQ ID NO:1或SEQ ID NO:2所示。
2.根据权利要求1所示的gRNA,其特征在于,所述启动子元件的序列如SEQ ID NO:3所示;所述骨架序列如SEQ ID NO:4所示。
3.根据权利要求1所示的gRNA,其特征在于,包括SEQ ID NO:5~6所示的两条gRNA。
4.靶向斑马鱼p2rx2基因的敲除试剂,其特征在于,包括权利要求1~3任一项所述的gRNA和Cas9 mRNA。
5.根据权利要求4所述的敲除试剂,其特征在于,敲除试剂中包括Nuclease free H2O和如下浓度的:
MgCl2 10mmol/L;
Cas9 mRNA 150ng/μL;
gRNA 各20ng/μL。
6.权利要求4或5所述的敲除试剂在构建p2rx2基因缺失型斑马鱼模型中的应用。
7.一种构建p2rx2基因缺失型斑马鱼模型的方法,其特征在于,将权利要求4或5所述的敲除试剂接入一细胞期的斑马鱼受精卵内,孵化获得p2rx2基因缺失型斑马鱼模型。
8.根据权利要求7所述的方法,其特征在于,每个受精卵给予1.5~2.0nL权利要求4或5所述的敲除试剂。
9.根据权利要求7或8所述的方法,其特征在于,所述孵育后还包括筛选的步骤;所述筛选包括:对受精36h后的胚胎进行筛选,或对成鱼进行筛选;
所述筛选包括对基因组DNA进行扩增后进行Sanger测序或根据电泳结果进行筛选;扩增产物包含227bp和322bp大小的条带则p2rx2基因缺失,扩增产物仅有322bp大小的片段则p2rx2基因未缺失;所述扩增的引物序列如SEQ ID NO:7~8所示。
10.根据权利要求7~9任一项所述的方法,其特征在于,所述孵化获得的p2rx2基因缺失斑马鱼为F0代;所述方法还包括传代的步骤;
F1代斑马鱼由F0代斑马鱼与野生型的斑马鱼杂交获得;
F2代斑马鱼由F1代突变体杂交获得。
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