CN107988268A - 一种基因敲除选育tcf25基因缺失型斑马鱼的方法 - Google Patents
一种基因敲除选育tcf25基因缺失型斑马鱼的方法 Download PDFInfo
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Abstract
本发明涉及基因敲除技术领域,特别是公开了一种基因敲除选育tcf25基因缺失型斑马鱼的方法,通过CRISPR/Cas9基因编辑技术,在斑马鱼的tcf25基因上设计合适的打靶位点,在体外合成的特异性sgRNA和Cas9‑mRNA,显微共注射进入斑马鱼一细胞内,胚胎培养60h后,通过选取胚胎进行基因型分析,从而证实了所选位点的有效性。本发明脱靶率很低,且干扰掉tcf25基因,并且通过遗传学手段研究其功能,有助于进一步揭示心脏形态发生的整个过程以及调控这些过程的分子机制,在医学上心脏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。
Description
技术领域
本发明涉及基因敲除领域,特别是公开了一种基因敲除选育tcf25基因缺失型斑马鱼的方法。
背景技术
tcf25(transcription factor 25)基因位于斑马鱼第18号染色体上,包含18个外显子和17个内含子,cDNA全长1920bp,编码645氨基酸,tcf25包含有3个进化上保守的功能结构域,研究发现,利用Morpholino干扰技术,干扰掉tcf25基因斑马鱼胚胎,出现明显的发育畸形,同时通过基因差异表达谱分析和基因组关联分析等,发现tcf25在人类胚胎早期的多个组织中都有表达,特别是心脏中强烈表达。
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,且tcf25基因进化上较为保守,研究发现tcf25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。
通过CRISPR/Cas9基因编辑技术,在斑马鱼的tcf25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60h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。
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 short palindromic 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,但同时该技术存在一定的缺陷,其脱靶率相对较高。
发明内容
本发明为了克服上述技术问题的不足,提供了一种基因敲除选育tcf25基因缺失型斑马鱼的方法,找到了合适的打靶位点,通过CRISPR/Cas9基因编辑技术,选育出tcf25基因缺失型斑马鱼。
解决上述技术问题的技术方案如下:
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tcf25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tcf25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tcf25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):
tgTAATACGACTCACTATAGGAAGCATGTCTGTCAGATGGTTTTAGAGCTAGAAATAGC
F2(靶位点b正向引物):
tgTAATACGACTCACTATAGAAGAAGAAGAAGAAAAATAGTTTTAGAGCTAGAAATAGC
R(共用的反向引物):AAGCACCGACTCGGTGCCACT
PCR检测引物
PCR检测引物上下游引物分别位于1号和3号内含子上:
F(5’-GACTATGGTGACTTGGGGGA-3’)
R(5’-TAATTCCATGTCAACAGCTCC-3
2)构建gRNA表达载体以及gRNA体外合成
a,首先将gRNA骨架克隆到p42250载体上,取1-2μL质粒进行琼脂糖凝胶电泳检测。
b,特异性gRNA体外合成
用BsaI限制性内切酶线性化此质粒。一般来说,酶切反应总体积为20μL,体系如下:
混匀后于37℃水浴,酶切2h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(50μL)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℃30s,延伸72℃30s)30个循环,再72℃8min。待反应结束后,离心PCR产物,取1μL样品点样于1.2%琼脂糖凝胶上进行电泳,凝胶成像***拍摄结果。
d,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。
e,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1μL DNA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1.2%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;
f,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Cas9mRNA和gRNA配成混合液,充分混匀,使Cas9mRNA的终浓度为150ng/μL,gRNA的终浓度为20ng/μL。注射约1.8nL Cas9mRNA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,挑选部分发育正常的早期胚胎,检测其tcf25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60小时后(60hpf),分别收集野生型(做对照)和注射后胚胎于1.5mL Ep管中(每管10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30μL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(20μL)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56℃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1.2%琼脂糖凝胶上进行电泳,检测PCR产物大小是否正确。
c、若PCR产物正确,则用1.2%琼脂糖凝胶电泳分离PCR产物,在紫外下切下目的条带,进行纯化回收。
d、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。
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538bp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400bp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538bp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)。
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538bp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tcf25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。
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。
本发明的有益效果是:
通过CRISPR/Cas9基因编辑技术,在斑马鱼的tcf25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60h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,脱靶率很低,且干扰掉fhl1b基因,并且通过遗传学手段研究其功能,有助于进一步揭示心脏形态发生的整个过程以及调控这些过程的分子机制,在医学上心脏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。
下面结合附图和具体实施方式对本发明作进一步详细的说明。
附图说明
图1为CRISPR/Cas9打靶***的原理图;
图2为tcf25基因上靶位点的结构图;
图3为斑马鱼F1代电泳结果图;
图4为缺失型和WT型基因序列正向对比;
图5为靶位点附近缺失对比;
具体实施方式
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tcf25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tcf25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tcf25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):
tgTAATACGACTCACTATAGGAAGCATGTCTGTCAGATGGTTTTAGAGCTAGAAATAGC
F2(靶位点b正向引物):
tgTAATACGACTCACTATAGAAGAAGAAGAAGAAAAATAGTTTTAGAGCTAGAAATAGC
R(共用的反向引物):AAGCACCGACTCGGTGCCACT
PCR检测引物
PCR检测引物上下游引物分别位于1号和3号内含子上:
F(5’-GACTATGGTGACTTGGGGGA-3’)
R(5’-TAATTCCATGTCAACAGCTCC-3
3)构建gRNA表达载体以及gRNA体外合成
a,首先将gRNA骨架克隆到p42250载体上,取1-2μL质粒进行琼脂糖凝胶电泳检测。
b,特异性gRNA体外合成
用BsaI限制性内切酶线性化此质粒。一般来说,酶切反应总体积为20
μL,体系如下:
混匀后于37℃水浴,酶切2h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(50μL)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℃30s,延伸72℃30s)30个循环,再72℃8min。待反应结束后,离心PCR产物,取1μL样品点样于1.2%琼脂糖凝胶上进行电泳,凝胶成像***拍摄结果。
d,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。
e,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1μL DNA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1.2%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;
f,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Cas9mRNA和gRNA配成混合液,充分混匀,使Cas9mRNA的终浓度为150ng/μL,gRNA的终浓度为20ng/μL。注射约1.8nL Cas9mRNA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,挑选部分发育正常的早期胚胎,检测其tcf25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60小时后(60hpf),分别收集野生型(做对照)和注射后胚胎于1.5mL Ep管中(每管10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30μL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(20μL)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56℃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1.2%琼脂糖凝胶上进行电泳,检测PCR产物大小是否正确。
c、若PCR产物正确,则用1.2%琼脂糖凝胶电泳分离PCR产物,在紫外下切下目的条带,进行纯化回收。
d、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。
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538bp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400bp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538bp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)。
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538bp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tcf25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。
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。
附图3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,2,4,6,8,9号泳道与野生型相比,除538bp的目的条带外,还有一条450bp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(538bp)进行对比,发现tcf25两个靶位点处(粗体表示,下划线)只有靶点a附近有碱基缺失,靶位点a处有48个碱基的缺失(缺失16个氨基酸)。由于筛选到的突变体的F1代的tcf25基因部分碱基缺失造成整个基因的移码突变,改变斑马鱼了tcf25基因的表达。从而影响斑马鱼的心脏的发育。
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。
Claims (6)
1.一种基因敲除选育tcf25基因缺失型斑马鱼的方法,其特征在于,包括以下步骤:
1)分别设计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tcf25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tcf25基因的靶位点;
两对特异性靶位点PCR引物如下:
F1(靶位点a正向引物):
tgTAATACGACTCACTATAGGAAGCATGTCTGTCAGATGGTTTTAGAGCTAGAAATAGC
F2(靶位点b正向引物):
tgTAATACGACTCACTATAGAAGAAGAAGAAGAAAAATAGTTTTAGAGCTAGAAATAGC
R(共用的反向引物):AAGCACCGACTCGGTGCCACT
PCR检测引物
PCR检测引物上下游引物分别位于2号和3号内含子上:
F(5’-GACTATGGTGACTTGGGGGA-3’)
R(5’-TAATTCCATGTCAACAGCTCC-3;
2)构建gRNA表达载体以及特异性gRNA体外合成
a,首先将gRNA骨架克隆到p42250载体上;
b,特异性gRNA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20μL,体系如下:
混匀后于37℃水浴,酶切2h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反应体系如下:
震荡混匀之后,4℃离心,于PCR仪上进行扩增反应;
d,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;
e,测定纯化的DNA浓度,再以此DNA为模板,用20μL体系进行体外转录,合成特异性gRNA;具体操作如下:
体外转录反应体系:
将反应物全部加入0.2mL EP管中,混匀之后,于37℃水浴2h;
水浴结束后,消化DNA模板,然后电泳;
f,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℃;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Cas9mRNA和gRNA配成混合液,充分混匀,使Cas9mRNA的终浓度为150ng/μL,gRNA的终浓度为20ng/μL,注射1.8nL Cas9mRNA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,挑选部分发育正常的早期胚胎,检测其tcf25基因是否存在突变;
a,提取斑马鱼基因组
斑马鱼胚胎受精60小时后(60hpf),分别收集野生型和注射后胚胎于1.5mL Ep管中,每管10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,加入等体积预先冷却的异丙醇于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30μL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后,根据CRISPR靶位点上下游约150-200bp的基因组区域,利用PrimerPremier 5.0软件设计引物序列以扩增出目的DNA片段;
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;
c,用1.2%琼脂糖凝胶电泳分离PCR产物,在紫外下切下目的条带,进行纯化回收;
d,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;
e,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;
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538bp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400bp左右的小带,如果此突变可以遗传到F1代,则PCR扩增会出现小于538bp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;
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538bp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tcf25突变体纯合子;挑选检验结果证明存在纯合子,养大后再单条剪尾鉴定;
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。
2.根据权利要求1所述的基因敲除选育tcf25基因缺失型斑马鱼的方法,其特征在于,步骤1)中靶位点的选择遵循以下标准: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;靶点的选择位置在基因的结构域内。
3.根据权利要求1所述的基因敲除选育tcf25基因缺失型斑马鱼的方法,其特征在于,步骤2)中所述的转录实验中所用Tip头,EP管均为DEPC处理过的RNase-Free。
4.根据权利要求1所述的基因敲除选育tcf25基因缺失型斑马鱼的方法,其特征在于,步骤2)的c步骤中所述的于PCR仪上进行扩增反应,其反应条件为:预变性95℃3min,再重复30次循环以下步骤:变性95℃15s----退火60℃30s---延伸72℃30s,再72℃8min;待反应结束后,离心PCR产物,进行电泳。
5.根据权利要求1所述的基因敲除选育tcf25基因缺失型斑马鱼的方法,其特征在于,步骤3)中所述的E3水为5mmol/L NaCl,0.33mmol/L CaCl2,0.33mmol/L MgSO4,0.17mmol/LKCl的混合物。
6.根据权利要求1所述的基因敲除选育tcf25基因缺失型斑马鱼的方法,其特征在于,步骤4)的b步骤中所述的于PCR仪上进行扩增反应,其反应条件为:预变性95℃ 5min,再重复30次循环以下步骤:变性95℃ 30s---退火56℃ 30s---延伸72℃ 30s,再72℃ 8min;待反应结束后,离心PCR产物,进行电泳。
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