CN116515825A - 一种敲除斑马鱼ddx18基因的sgRNA组合及其应用 - Google Patents
一种敲除斑马鱼ddx18基因的sgRNA组合及其应用 Download PDFInfo
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Abstract
本发明提供了一种敲除斑马鱼ddx18基因的sgRNA组合及其应用,属于基因敲除技术领域。本发明提供了一种sgRNA组合,所述sgRNA组合包括第一sgRNA、第二sgRNA、第三sgRNA和第四sgRNA。通过CRISPANT技术,本发明的sgRNA组合能够特异性敲除斑马鱼ddx18基因第5号和6号外显子上177bp的基因片段,选育出ddx18基因缺失型斑马鱼,从而构建斑马鱼白血病模型。本发明提供的斑马鱼白血病模型为进一步开展ddx18基因突变与白血病发病机制的关系研究及筛选抗白血病药物奠定了很好的基础。
Description
技术领域
本发明属于基因敲除技术领域,具体涉及一种敲除斑马鱼ddx18基因的sgRNA组合及其应用。
背景技术
CRISPR-Cas基因编辑技术是对特定位点进行基因编辑的技术,利用CRISPR-Cas技术在某一特定位点导入目的基因,可以使有基因缺陷的细胞恢复正常功能,也可以在正常细胞内导入疾病相关基因从而构建相应疾病模型。研究人员发现使用多个sgRNA靶向同一基因,可以最大限度地提高编辑效率,并将这种方法命名为CRISPANT技术。血液系统疾病多半由于基因突变引起。利用CRISPANT技术可研究人类血液疾病的遗传背景,矫正致病的突变基因,发现更多与血液疾病预后相关的基因,有望治愈血液恶性疾病;此外,还可构建更接近人体生理特性的个体化血液疾病模型,也有利于进行新药开发试验。
DEAD-box RNA解旋酶18(DDX18)是DDX家族的一个高度保守成员,该家族是一类以D(Asp)-E(Glu)-A(Ala)-D(Asp)为保守序列的RNA解旋酶家族,参与RNA相关的细胞活动,包括核糖体生物合成、microRNA加工和mRNA转运。目前,越来越多的研究表明,DDX家族在肿瘤组织中异常表达,其表达水平与肿瘤的生长、转移及预后相关,并且表现出促癌和抑癌的双向作用。虽然这些成员在肿瘤发生中的确切作用尚未明确,但可能与RNA解旋酶的正常功能失调而导致异常RNA翻译所形成的关键蛋白表达和功能有关。有研究表明DDX18在斑马鱼原始造血的细胞周期进展中起重要作用,ddx18基因突变已在血液系统恶性肿瘤患者中发现。前期研究发现,ddx18缺失突变体斑马鱼外周血细胞的原始粒细胞和幼稚粒细胞明显增多。初步表明ddx18基因突变体的血液髓系细胞成熟过程受阻虽然DDX家族成员与许多恶性肿瘤相关,但DDX18与白血病发生发展的相关性,以及与白血病干细胞/前体细胞的增殖与分化的关系研究较少。通过构建突变体模型,可为进一步探讨ddx18对造血系统的作用机制提供实验材料。
目前ddx18基因在斑马鱼中的作用研究主要方法是Morpholino介导的基因敲降方法。该技术方法虽然可快速研究目的基因功能,但也存在缺点,即该技术在mRNA水平降低基因表达,仍有基因表达产物发挥作用,某些特殊的表型不易被观察;再者该技术只能短期降低斑马鱼体内目的基因的表达,无法实现长期观察由于基因表达缺失造成的功能障碍。
发明内容
有鉴于此,本发明的目的在于提供一种敲除斑马鱼ddx18基因的sgRNA组合及其应用,本发明的基因敲除的DNA位点能在斑马鱼稳定遗传,表型观察不受时间限制。
本发明提供了一种sgRNA组合,所述sgRNA组合包括第一sgRNA、第二sgRNA、第三sgRNA和第四sgRNA;所述第一sgRNA包括核苷酸序列如SEQ ID NO.1所示的第一正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物;所述第二sgRNA包括核苷酸序列如SEQ IDNO.2所示的第二正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物;所述第三sgRNA包括核苷酸序列如SEQ ID NO.3所示的第三正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物;所述第四sgRNA包括核苷酸序列如SEQ ID NO.4所示的第四正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物。
本发明还提供了上述方案所述的sgRNA组合在CRISPANT特异性敲除斑马鱼ddx18基因构建斑马鱼白血病模型中的应用。
优选的,所述敲除斑马鱼ddx18基因是敲除斑马鱼ddx18基因第5号和6号外显子上177bp的基因片段;所述基因片段的核苷酸序列如SEQ ID NO.8所示。
本发明还提供了一种用于构建斑马鱼白血病模型的试剂盒,包括上述方案所述的sgRNA组合和Cas9蛋白。
本发明还提供了一种斑马鱼白血病模型的构建方法,包括如下步骤:
将上述方案所述的sgRNA组合和Cas9蛋白混合物注射到斑马鱼的受精卵中,受精卵发育成胚胎;
利用检测引物组从所述胚胎中PCR筛选出敲除有效的胚胎,培养至成鱼,获得F0代突变斑马鱼;
将F0代突变斑马鱼与野生型斑马鱼杂交得到F1代胚胎,利用检测引物组PCR筛选出存在突变的胚胎,培养至成鱼,筛选得到可遗传的斑马鱼突变体的F1代。
优选的,所述检测引物组包括核苷酸序列如SEQ ID NO.6所示的上游引物和核苷酸序列如SEQ ID NO.7所示的下游引物。
优选的,所述PCR的扩增体系以20μl计,包括以下组分:2×Mastermix10μl、超纯水7μl、上游引物1μl、下游引物1μl和基因组DNA模板1μl。
优选的,所述PCR的扩增程序为:95℃、3min;95℃、30s,56℃、30s,72℃、50s,35个循环;72℃、10min。
本发明还提供了上述方案所述构建方法构建得到的斑马鱼白血病模型在筛选抗白血病药物中应用。
本发明提供了一种sgRNA组合,所述sgRNA组合包括第一sgRNA、第二sgRNA、第三sgRNA和第四sgRNA;所述第一sgRNA包括核苷酸序列如SEQ ID NO.1所示的第一正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物;所述第二sgRNA包括核苷酸序列如SEQ IDNO.2所示的第二正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物;所述第三sgRNA包括核苷酸序列如SEQ ID NO.3所示的第三正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物;所述第四sgRNA包括核苷酸序列如SEQ ID NO.4所示的第四正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物。通过CRISPANT技术,本发明的sgRNA组合能够特异性敲除斑马鱼ddx18基因第5号和6号外显子上177bp的基因片段,选育出ddx18基因缺失型斑马鱼,从而构建斑马鱼白血病模型。
本发明使用多个sgRNA靶向同一基因,可以最大限度地提高编辑效率,更有效进行基因编辑,在目标基因DNA水平的随机插入或缺失多种核酸序列,产生每个个体中存在不同基因型的细胞,揭示基因功能;基因敲除的DNA位点能在斑马鱼稳定遗传,表型观察不受时间限制,可快速大批量进行基因筛选。
本发明提供的构建方法构建得到的斑马鱼白血病模型为进一步开展ddx18基因突变与白血病发病机制的关系研究及筛选抗白血病药物奠定了很好的基础。
附图说明
图1为体外合成制备的sgRNA质量电泳鉴定结果图,自左向右第5泳道为DNAMarker:从下向上依次为100bp、250bp、500bp、750bp、1000bp、2000bp;泳道1~4依次为第一sgRNA、第二sgRNA、第三sgRNA、第四sgRNA;
图2为候选F1基因组DNA片段PCR扩增产物电泳结果图;结果参见图1,其中,自左向右第5泳道为BM5000 DNAMarker:从下向上依次为100bp、250bp、500bp、750bp、1000bp、2000bp、3000bp、5000bp;其余按自左向右泳道分别为编号1~4号斑马鱼尾鳍组织PCR产物电泳结果;图1中1~4泳道扩增产物大小均为316bp;
图3为携带(-66+21bp)移码突变F1斑马鱼测序峰图,上图红色方框显示自226bp开始出现(-66+21bp)移码突变;
图4为携带(+3–2bp)移码突变F1斑马鱼测序峰图,上图红色方框显示自79bp开始出现(+3–2bp)移码突变。
具体实施方式
本发明提供了一种sgRNA组合,所述sgRNA组合包括第一sgRNA、第二sgRNA、第三sgRNA和第四sgRNA;所述第一sgRNA包括核苷酸序列如SEQ ID NO.1所示的第一正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物;所述第二sgRNA包括核苷酸序列如SEQ IDNO.2所示的第二正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物;所述第三sgRNA包括核苷酸序列如SEQ ID NO.3所示的第三正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物;所述第四sgRNA包括核苷酸序列如SEQ ID NO.4所示的第四正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物。
在本发明中,SEQ ID NO.1所示核苷酸序列具体为:
在本发明中,SEQ ID NO.2所示核苷酸序列具体为:
在本发明中,SEQ ID NO.3所示核苷酸序列具体为:
在本发明中,SEQ ID NO.4所示核苷酸序列具体为:
其中,SEQ ID NO.1~SEQ ID NO.4所示核苷酸序列中,加粗序列为T7启动子部分,下划线序列为CRISPR序列,小写字母为部分sgRNA骨架模板序列。
在本发明中,所述反向引物为通用引物R-Common,核苷酸序列如SEQ ID NO.5所示,具体为:AAAAAAAGCACCGACTCGGTGCCAC。
在本发明中,第一sgRNA、第二sgRNA、第三sgRNA和第四sgRNA对应的结合位点的核苷酸序列分别如SEQ ID NO.10~SEQ ID NO.13所示;SEQ ID NO.10所示核苷酸序列具体为:CATCATGGGTGGCAGCAATC;SEQ ID NO.11所示核苷酸序列具体为:GAAGCCCAGAAACTCGCTAA;SEQ ID NO.12所示核苷酸序列具体为:GGCTTCATGTTTAAAAACCT;SEQ ID NO.13所示核苷酸序列具体为:GCTGACAGGATTCTGGAGGT。
本发明还提供了上述方案所述的sgRNA组合在CRISPANT特异性敲除斑马鱼ddx18基因构建斑马鱼白血病模型中的应用。
在本发明中,所述斑马鱼ddx18基因优选为斑马鱼9号染色体ddx18基因第5号和6号外显子上长度为316 bp的基因(序列编号为NM 001003411.1,参见图1),核苷酸序列如SEQ ID NO.8所示,具体为:
其中加粗序列位于第5号外显子上,未加粗序列位于第6号外显子上;
所述斑马鱼ddx18基因更优选为斑马鱼ddx18基因第5号和6号外显子上长度为177bp的基因片段,所述基因片段的核苷酸序列如SEQ ID NO.9所示,具体为:
本发明还提供了一种用于构建斑马鱼白血病模型的试剂盒,包括上述方案所述的sgRNA组合和Cas9蛋白。
本发明还提供了一种斑马鱼白血病模型的构建方法,包括如下步骤:
将上述方案所述的sgRNA组合和Cas9蛋白混合物注射到斑马鱼的受精卵中,受精卵发育成胚胎;
利用检测引物组从所述胚胎中PCR筛选出敲除有效的胚胎,培养至成鱼,获得F0代突变斑马鱼;
将F0代突变斑马鱼与野生型斑马鱼杂交得到F1代胚胎,利用检测引物组PCR筛选出存在突变的胚胎,培养至成鱼,筛选得到可遗传的斑马鱼突变体的F1代。
本发明首先将上述方案所述的sgRNA组合和Cas9蛋白混合物注射到斑马鱼的受精卵中,受精卵发育成胚胎。
在本发明中,所述sgRNA组合的工作浓度优选为400 ng/μl;所述第一sgRNA、第二sgRNA、第三sgRNA、第四sgRNA的浓度比优选为1:1:1:1;所述Cas9蛋白的终浓度优选为400ng/μl。
受精卵发育成胚胎后,本发明利用检测引物组从所述胚胎中PCR筛选出敲除有效的胚胎,培养至成鱼,获得F0代突变斑马鱼。
获得F0代突变斑马鱼后,本发明将F0代突变斑马鱼与野生型斑马鱼杂交得到F1代胚胎,利用检测引物组PCR筛选出存在突变的胚胎,培养至成鱼,筛选得到可遗传的斑马鱼突变体的F1代。
在本发明中,所述检测引物组优选的包括核苷酸序列如SEQ ID NO.6所示的上游引物和核苷酸序列如SEQ ID NO.7所示的下游引物。
在本发明中,所述PCR的扩增体系以20μl计,优选的包括以下组分:2×Mastermix10μl、超纯水7μl、上游引物1μl、下游引物1μl和基因组DNA模板1μl。
在本发明中,所述PCR的扩增程序优选为:95℃、3min;95℃、30s,56℃、30s,72℃、50s,35个循环;72℃、10min。
本发明还提供了上述方案所述构建方法构建得到的斑马鱼白血病模型在筛选抗白血病药物中应用。
下面将结合本发明中的实施例,对本发明中的技术方案进行清楚、完整地描述。
实施例1Crispant策略敲降斑马鱼ddx18基因突变体的构建方法,包括以下步骤:
步骤一:Crispant策略敲降靶位点设计和PCR检测引物
在NCBI数据库上查询斑马鱼ddx1 8基因的基因组DNA序列,并分析功能结构域,设计斑马鱼ddx1 8基因的CRISPR序列,位于基因组的第5,6号外显子上;为了最大限度地提高编辑效率,更有效进行基因编辑,设计并选择4个sgRNA结合位点。
步骤二:目标基因的基因型确认
2.1 PCR扩增的引物设计
表1 PCR扩增的引物
序列名称 | 序列 | 用途 |
ddx18-F1 | ATAGTTCTGTCTCCTACACG,SEQ ID NO.6 | 正向扩增引物 |
ddx18-R1 | ATCACTGCTTCTTACTAGG,SEQ ID NO.7 | 反向扩增引物 |
2.2基因组DNA模板制备
2.2.1将野生型斑马鱼尾鳍组织置于200μl PCR管内,向PCR管中加入基因组提取试剂盒buffer 10μl。
2.2.2制备基因组DNA模板的反应条件:65℃、30min,95℃、5min,16℃、1min,4℃。
2.3PCR反应体系及条件
2.3.1PCR反应体系(20μl)
2×Mastermix 10μl、7μl的超纯水、1μl浓度为5μM的ddx18-F1、1μl浓度为5μM的ddx18-R1和1μl的由2.2步骤获得的基因组DNA模板。
2.3.2PCR反应条件
95℃、3min,35×(95℃、30s,56℃、30s,72℃、50s),72℃、10min,4℃。
2.4PCR产物电泳结果
取2μl进行琼脂糖凝胶电泳(1%)鉴定其完整性,候选F1基因组DNA片段PCR扩增产物均为316bp。
步骤三:PCR产物进行sgRNA体外合成
3.1sgRNA模板的制备(PCR法)
3.1.1sgRNA表达构件体外合成引物
3.1.2PCR反应体系
2×Mastermix 40μl、35μl的超纯水、2μl的正、反向引物(5μM)和1μl的pYSY-sgRNA质粒(10ng/μl)。其中正向引物为ddx18-sgRNA(1~4)-F,ddx18-sgRNA(1~4)-F的核苷酸序列分别如SEQ ID NO.1~4所示,反向引物为R-Common,核苷酸序列如SEQ ID NO.5所示。
3.1.3PCR反应条件
95℃、3min,35×(95℃、30s,56℃、30s,72℃、30s),72℃、10min,4℃。
3.1.4PCR产物纯化(无核酸酶污染处理)
将上述PCR产物分别利用无核酸酶污染的PCR clean up(Axygen)试剂盒收集。回收溶剂为无核酸酶污染的超纯水。
3.2sgRNA的体外转录
3.2.1体外转录过程
将步骤3.1所得到的纯化后模板分别用T7 RNA聚合酶进行体外转录。使用RNA体外转录试剂盒(MAXIscriptT7,Ambion,USA),按试剂盒说明书要求依次加入10×Transcription Buffer4μl、10mMATP 2μl、10mM CTP2μl、10mM GTP 2μl、10mM UTP 2μl、T7RNApolymerase mix 4μl、步骤3.1得到的sgRNA模板DNA24μl,轻弹混匀离心后,37℃水浴3h。
加入DNase I(Ambion,USA)1.5μl,37℃水浴15min,以去除模板。
之后加入160μl DEPC水扩大体积至200μl,同时加入20μl无核酸酶3M醋酸钠(pH5.2)和3倍体积的无水乙醇(生工),-80℃沉淀过夜。4℃12,000g离心20min,去除上清后,加入无核酸酶75%乙醇,4℃12,000g离心20min,去除上清后,沉淀在通风橱晾干,然后以20μl无核酸酶超纯水重悬后储存于-80℃冰箱备用。
3.2.2sgRNA质量鉴定
将sgRNA取1μl进行琼脂糖凝胶电泳(1%)鉴定其完整性,图1为sgRNA质量电泳鉴定结果图。自左向右第5泳道为DNAMarker:从下向上依次为100bp、250bp、500bp、750bp、1000bp、2000bp;泳道1~4依次为第一sgRNA、第二sgRNA、第三sgRNA、第四sgRNA。
步骤四:斑马鱼受精卵的显微注射
按常规方法收取斑马鱼受精卵。将上述sgRNA分别和Cas9蛋白(全部sgRNA总的终浓度为400ng/μl,第一sgRNA、第二sgRNA、第三sgRNA、第四sgRNA的浓度比为1:1:1:1;Cas9蛋白终浓度为400ng/μl)混合后,显微注入斑马鱼受精卵,注射量为1nl每胚胎。
步骤五:sgRNA引导Cas9靶向切割目标基因组DNA序列效率的确认。
5.1目标基因组DNA模板的制备
待注射胚胎发育至24hpf(hourpost-fertilization)时,每组分别取16枚胚胎单胚胎制备基因组DNA模板,使用斑马鱼基因型鉴定试剂盒制备基因组DNA模板,具体反应条件为:65℃、30min,95℃、5min,16℃、1min,4℃。
5.2PCR反应体系(20μl)
2×Mastermix10μl、7μl的超纯水、1μl浓度为5μM的ddx18-F1、1μl浓度为5μM的ddx18-R1和1μl的基因组DNA模板;
5.3PCR反应条件
PCR反应的条件为:95℃、3min,35×(95℃、30s,56℃、30s,72℃、50s),72℃、10min,4℃。
5.4扩增产物的测序
上述PCR产物经过电泳确认条带大小后,将阳性扩增产物全部送商业公司测序。
步骤六:Crispant策略敲降斑马鱼ddx18基因F0代的可遗传鉴定
将性成熟的预计靶向敲除斑马鱼ddx18基因的F0代雌雄分开,等待1周后,分别与Tuebingen(TU)斑马鱼进行1对1交配。待获得的1号、2号候选阳性F0与TU野生型斑马鱼的后代胚胎发育至24hpf时,每一组随机挑取16枚胚胎,分别进行基因型鉴定。
鉴定出ddx18基因组编辑突变体1号2号为阳性可遗传F0,将1号2号交配繁育F1,用于后续突变体筛选。
步骤七:候选F1突变体斑马鱼的繁育与饲养
将注射了Cas9和sgRNA的F0胚胎饲养至性成熟,然后令其自由交配,获得F1胚胎。将F1胚胎按常规饲养至成熟,进行基因型鉴定,确定携带可种系遗传突变等位基因的阳性F0。将筛选出的阳性F0斑马鱼雌雄交配,然后将F1胚胎按常规饲养至2月龄以上,进行基因型鉴定,以筛选基因编辑突变体。
7.1候选F1突变体基因组DNA模板制备
7.1.1组织取材
取3~4月龄的F1成鱼,剪取部分尾鳍组织,按顺序分别放入200μl PCR管中,然后置于冰上存放。
7.1.2基因组模板DNA制备
向含有尾鳍组织的200μl PCR管中加入的YSYbuffer 10μl。快速离心后,将PCR管放入PCR仪中,进行如下反应:65℃、30min,95℃、5min,16℃、1min,4℃。
7.2基因组DNA片段PCR扩增
7.2.1反应的组成
反应体系:
2×Mastermix10μl、7μl的超纯水、1μl的正反向(ddx18-F1,ddx18-R1)引物(5μM)和1μl的基因组DNA模板;
7.2.2PCR反应条件
PCR反应的条件为:95℃3min,35×(95℃30s,56℃30s,72℃50s),72℃10min,4℃。
7.3携带ddx18突变等位基因的F1突变体的筛选鉴定
选取4尾(编号1~4)斑马鱼的基因组DNA进行PCR,结果参见图1,PCR产物分别送至商业公司直接进行Sanger测序。
共筛选到ddx18移码杂合突变斑马鱼(F1)2尾,2种突变体。结果参见表2~表4及图3和图4,测序图谱显示斑马鱼编号:3、4出现双峰,且在PAM位点附近(如方框内),其与野生型相比,分别使斑马鱼ddx18第六个外显子产生-66+21移码突变(与原始序列相比缺失了66个碱基,又增加了21个碱基)、第六个外显子产生+3–2bp移码突变(与原始序列相比增加了3个碱基,又缺失了2个碱基)。
表2 F1代ddx18突变斑马鱼基因型测序鉴定结果
斑马鱼编号 | 突变类型 |
3 | -66+21bp |
4 | +3–2bp |
表3(-66+21bp)移码与野生型等位基因比对结果
表4(+3-2bp)移码与野生型等位基因比对结果
上述步骤中sgRNA体外合成和sgRNA引导Cas9靶向切割目标基因组DNA序列效率的确认是核心步骤,使用多个sgRNA靶向同一基因,可以最大限度地提高编辑效率,决定了对于ddx18基因靶点序列产生多种核酸序列的随机插入或缺失的效率,对于构建突变体起着决定性的作用。
尽管上述实施例对本发明做出了详尽的描述,但它仅仅是本发明一部分实施例而不是全部实施例,人们还可以根据本实施例在不经创造性前提下获得其他实施例,这些实施例都属于本发明保护范围。
Claims (9)
1.一种sgRNA组合,其特征在于,所述sgRNA组合包括第一sgRNA、第二sgRNA、第三sgRNA和第四sgRNA;
所述第一sgRNA包括核苷酸序列如SEQ ID NO.1所示的第一正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物;
所述第二sgRNA包括核苷酸序列如SEQ ID NO.2所示的第二正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物;
所述第三sgRNA包括核苷酸序列如SEQ ID NO.3所示的第三正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物;
所述第四sgRNA包括核苷酸序列如SEQ ID NO.4所示的第四正向引物和核苷酸序列如SEQ ID NO.5所示的反向引物。
2.权利要求1所述的sgRNA组合在CRISPANT特异性敲除斑马鱼ddx18基因构建斑马鱼白血病模型中的应用。
3.根据权利要求2所述的应用,其特征在于,所述敲除斑马鱼ddx18基因是敲除斑马鱼ddx18基因第5号和6号外显子上177bp的基因片段;所述基因片段的核苷酸序列如SEQ IDNO.8所示。
4.一种用于构建斑马鱼白血病模型的试剂盒,其特征在于,包括权利要求1所述的sgRNA组合和Cas9蛋白。
5.一种斑马鱼白血病模型的构建方法,其特征在于,包括如下步骤:
将权利要求1所述的sgRNA组合和Cas9蛋白混合物注射到斑马鱼的受精卵中,受精卵发育成胚胎;
利用检测引物组从所述胚胎中PCR筛选出敲除有效的胚胎,培养至成鱼,获得F0代突变斑马鱼;
将F0代突变斑马鱼与野生型斑马鱼杂交得到F1代胚胎,利用检测引物组PCR筛选出存在突变的胚胎,培养至成鱼,筛选得到可遗传的斑马鱼突变体的F1代。
6.根据权利要求5所述的构建方法,其特征在于,所述检测引物组包括核苷酸序列如SEQ ID NO.6所示的上游引物和核苷酸序列如SEQ IDNO.7所示的下游引物。
7.根据权利要求5所述的构建方法,其特征在于,所述PCR的扩增体系以20μl计,包括以下组分:2×Mastermix10μl、超纯水7μl、上游引物1μl、下游引物1μl和基因组DNA模板1μl。
8.根据权利要求5所述的构建方法,其特征在于,所述PCR的扩增程序为:95℃、3min;95℃、30s,56℃、30s,72℃、50s,35个循环;72℃、10min。
9.权利要求5~8任意一项所述构建方法构建得到的斑马鱼白血病模型在筛选抗白血病药物中应用。
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