CN110396525A - Rnf213基因完整敲除纯合子斑马鱼的制备方法和用途 - Google Patents
Rnf213基因完整敲除纯合子斑马鱼的制备方法和用途 Download PDFInfo
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Abstract
本发明公开了一种rnf213a基因完整敲除斑马鱼突变体的制备及其应用;设计针对斑马鱼rnf213a基因的TALEN序列;构建其左右臂质粒,并体外转录出相应的mRNA;将TALEN左右臂mRNA混合并注射到斑马鱼胚胎中;注射后的胚胎养至F0成鱼与野生型成鱼交配,将携带有rnf213a基因突变的胚胎养至F1成鱼;F1代鱼剪尾基因型鉴定发现存在两种杂合子基因型:+2bp及‑7bp;将杂合子与野生型成鱼外交3代,净化其遗传背景后,再将同种基因型的杂合子杂交得到纯合子两种基因型:+2bp及‑7bp。纯合子与纯合子杂交得到的胚胎进行表型观察发现存在与人相似的烟雾病表型及海绵状血管瘤表型。本发明通过TALEN基因编辑技术,能够快速高效的建立rnf213a基因完整敲斑马鱼品系,利用该模型能够开展针对烟雾病或/和海绵状血管瘤的药物筛选工作。
Description
技术领域
本发明属于基因工程技术领域,尤其涉及利用基因打把技术敲除斑马鱼rnf213a基因的方法。
背景技术
RNF213基因位于17号染色体,编码包含5207个氨基酸的环指蛋白,该基因有两个重要的功能区RING finger domain及AAA+ATPases domain。近些年,RNF213引起关注主要是因为研究发现RNF213是东亚人群烟雾病的易感基因,尤其是日本人;后期的研究还发现该基因位点突变p.R4810K还与亚洲人群颅内大动脉狭窄有关;欧洲的一项研究发现RNF213不同位点突变还与高加索人动脉瘤相关。RNF213基因可能与多种脑血管的发病机制相关。
RNF213 p.R4810K与烟雾病相关存在明显种族差异,主要集中在亚洲人,日本烟雾病病人该位点突变率为90.1%,韩国78.9%,中国23.1%;正常人也存在该位点的变异,日本人2.5%,韩国人2.7%,中国人0.9%。相对于日本及韩国,中国汉族烟雾病病人此突变位点突变率较低。高加索人烟雾病发病率是日本人的1/10,高加索人未发现RNF213 p.R4810K与烟雾病明显相关,这可能是高加索人烟雾病发病率较低的原因之一。在RNF213 p.R4810K与相同环境不同血统烟雾病病人的研究中发现,p.R4810K在56%亚洲血统烟雾病病人中存在,非亚洲血统烟雾病病人中无p.R4810K点突变。这提示RNF213 p.R4810K是否参与烟雾病的发病机制存在种族差异。
在转化医学的研究中,斑马鱼rnf213基因knockdown出现部分血管芽生,类似烟雾病的血管芽生;利用cre-loxp***敲除小鼠Rnf213基因32号外显子未模拟烟雾病的表型;对应人RNF213c.14429G>A点突变小鼠也未模拟烟雾病的表型。RNF213是否参与了烟雾病的发病机制尚存在争议。
近些年发现利用morpholino技术敲减相关基因容易出现表型,但表型在突变体中并不能重复,研究表明morpholino技术容易出现脱靶敲除其他基因从而造成表型特异性较差。
发明内容
本发明的目的在于克服上述现有技术的不足之处而提供利用TALEN技术完整敲除斑马鱼rnf213a基因,设计的靶点为斑马鱼rnf213a的2号外显子,并且通过外交3代再内交形成纯合子斑马鱼。通过观察纯合子突变体的表型,可避免注射及morpholino脱靶引起的假表型,增加表型的特异性。
为实现上述目的,本发明采取的技术方案为:
一种RNF213基因完整敲除纯合子斑马鱼的制备方法,包括以下步骤:
S1、设计针对斑马鱼RNF213基因2号外显子靶序列的TALEN序列;
S2、构建TALEN左、右臂质粒,并分别体外转录出TALEN左、右臂mRNA;
S3、将TALEN左、右臂mRNA混合并注射到转基因斑马鱼胚胎中,提取胚胎DNA测序发现TALEN的mRNA能作用于rnf213a基因2号外显子;
S4、将注射后的胚胎培养至F0代,并与野生型成鱼交配,获得F1代成鱼;
S5、鉴定F1是否存在rnf213a基因突变,并经DNA测序鉴定rnf213a基因突变类型,将含有rnf213a基因突变的杂合子与野生型外交3代,净化其遗传背景;
S6、将同种基因型的杂合子杂交得到纯合子两种基因型:+2bp及-7bp。
优选地,所述步骤S1中的斑马鱼RNF213基因2号外显子靶序列为SEQ ID NO.1所示。
优选地,所述步骤S1中的TALEN序列的左臂序列如SEQ ID NO.2所示;右臂序列如SEQ ID NO.3所示。
优选地,所述步骤S1中的TALEN序列还包括能识别不同碱基的核苷酸序列及其编码的氨基酸序列,其中识别碱基A的核苷酸序列如SEQ ID NO.4所示,其编码的氨基酸序列如SEQ ID NO.5所示;识别碱基T的核苷酸序列如SEQ ID NO.6所示,其编码的氨基酸序列如SEQ ID NO.7所示;识别碱基C的核苷酸序列如SEQ ID NO.8所示,其编码的氨基酸序列如SEQ ID NO.9所示;识别碱基G的核苷酸序列如SEQ ID NO.10所示,其编码的氨基酸序列如SEQ ID NO.11所示。
优选地,所述步骤S2中还包括将构建的质粒转化低拷贝的感受态细胞,将其扩大培养后,提取质粒,经限制性内酶切DNA测序鉴定,确定质粒DNA序列。
优选地,所述针对质粒测序设计的正向引物如SEQ ID NO.12所示;反向引物如SEQID NO.13所示。
优选地,所述步骤S5中经鉴定含有rnf213a基因突变的成鱼F1杂合子为+2bp突变体及-7bp突变体.
优选地,所述步骤S6中纯合子分别为+2bp突变体及-7bp突变体。
本发明还提供了一种根据所述的RNF213基因敲除斑马鱼突变体纯合子的制备方法建立的RNF213基因敲除斑马鱼突变体纯合子模型在作为针对烟雾病或/和海绵状血管瘤的药物筛选模型中的用途。
本发明的有益效果:本发明通过TALEN基因编辑技术完整敲除斑马鱼rnf213a基因,相对于CRISPR/Cas9***基因敲除脱靶概率较低。rnf213a突变体一般存在转基因背景Tg(flk1:eGFP/gata1:DsRed),具有红绿荧光,例如:红色标记红细胞,绿色标记血管内皮,但是通过本发明外交3代再内交获得的纯合子突变体,降低了遗传背景。本发明得到了rnf213a完整敲除的两种基因型,表型可以同时在两种基因型验证,增加了表型的特异性。本发明构建的rnf213a基因突变体可作为烟雾病及海绵状血管瘤的斑马鱼动物模型。
附图说明
图1为本发明F1代+2bp与-7bp突变体的测序结果(其中A为TALEN2号外显子核苷酸序列以及靶点;B为+2bp突变体测序结果;C为-7bp突变体测序结果);
图2为通过激光片层显微镜及单光子显微镜观察rnf213a-/-表型的观察结果(其中A-C表示+2bp、-7bp突变体表型类似烟雾病;D-F表示+2bp、-7bp突变体表型类似人的海绵状血管瘤)。
具体实施方式
为了更加简洁明了的展示本发明的技术方案、目的和优点,下面结合具体实施例及其附图对本发明做进一步的详细描述。
实施例
1、TALEN的靶点设计
(1)在Pubmed数据库获取斑马鱼rnf213a基因2号外显子的序列。按照TALEN设计原则,利用TALEN序列专业设计软件,针对rnf213a基因2号外显子进行设计。共有8种TALEN骨架载体,左右臂各4个,它们均含有NLS,N端,C端以及FokⅠ等基本结构。该系列载体中,预先组装了TALE重复结构域中,3’端的最后0.5个重复单元,又称为半位点(Half-site)。可特异性地识别TALEN靶点的最后一个碱基。根据识别A/T/C/G碱基的不同,分为左臂L1/L2/L3/L4,右臂L1/L2/L3/L4。
(2)针对斑马鱼rnf213a基因2号外显子设计的TALEN左臂序列为:5’-CTGCAGTGAGTGTGGACAT-3’,TALEN右臂序列:5’-TACGAGACAACACAAGGT-3’;斑马鱼RNF213基因2号外显子的靶序列为:5’-TAAACTGCAATCTCAA-3’。
2、TALEN表达载体的构建
(1)根据模块串联组装原理,采用TALEN试剂盒(上海斯丹赛生物有限公司),完成TALEN表达载体的构建,具体步骤为:将TALEN左右臂识别序列最后一位T去除,以一个或两个碱基组合作为一个模块,首个标记为1,最后一个标记为9,依次类推进行标记。
(2)取出相应编号的9个模块,每个模块取1.5ul,并将其加入同一个PCR管中。共有8种TALEN骨架载体,左右臂各4个,它们均含有NLS,N端,C端以及FokⅠ等基本结构。该系列载体中,预先组装了TALE重复结构域中,3’端的最后0.5个重复单元,又称为半位点(Half-site)。可特异性地识别TALEN靶点的最后一个碱基。根据识别A/T/C/G碱基的不同,分为左臂L1/L2/L3/L4,右臂R1/R2/R3/R4,结构如表1所示。
表1:左臂L1/L2/L3/L4和右臂R1/R2/R3/R4的结构示意
(3)按表2反应体系加样,先加入相应的TALEN骨架载体,然后将溶液3、溶液1、溶液2依次加入PCR管中,最后加水补至20ul,混匀、轻甩。
表2:左、右臂TALEN连接反应体系
上述反应条件如下
(4)取出PCR管并加入溶液4 1ul、溶液5 0.5ul(溶液5请37℃预先融化),混匀,在PCR仪或水浴锅中37℃孵育一小时。
(5)将最终产物转化含低拷贝感受态细胞的EP管中,混匀,在冰上放置30min,然后放入42℃水浴锅中热激45s,再迅速放置于冰上3min。向EP管中加入500ulSOC溶液,置于37℃,250rpm的摇床中培养30min。取出EP管后,在4000r/min的离心机中离心5min。弃去EP管中的大部分上清,留下约100ul,并将沉淀轻轻吹打混匀,均匀涂布于卡纳霉素抗性的平板中,倒置于37℃培养箱中过夜培养12-16小时。
3、质粒提取
(1)采用质粒小量提取试剂盒进行操作,挑取培养板中单菌落,接种到含有卡那霉素的5ml LB培养基中,37℃恒温摇床250rpm振荡过夜培养。
(2)为降低重组率,本研究选用低拷贝的感受态细菌。提取质粒时,每个克隆至少抽提4ml菌液。将菌液倒入1.5ml的EP管后,4℃下12000rpm离心30秒,收取沉淀。倒去上清,加入250ul预冷的溶液P1,在旋涡振荡器振荡,直至菌体沉淀完全打散为止。加入250ul溶液P2,温和颠倒离心管5次,混合内容物,切勿振荡。加350ul预冷的溶液P3,反复颠倒数次,可见白色絮状物出现。4℃下12000rpm离心10分钟,弃沉淀。将上清转移到吸附柱CP3中,静置10分钟。4℃12000rpm离心1分钟,倒掉收集管中的废液。向吸附柱中加入500l去蛋白液PD,12000rpm离心30-60sec,倒掉收集管中的废液,将吸附柱CP3放回收集管中。向吸附柱CP3加入600l漂洗液PW,12,000rpm离心30-60sec,倒掉收集管中的废液,将吸附柱CP3放入收集管中,重复洗涤1次。最后12,000rpm离心2min,将吸附柱中残余的漂洗液去除。将吸附柱CP3开盖,置于室温放置数分钟,晾干吸附柱中残余的漂洗液。将其置于干净的离心管中,向吸附膜中间滴加50-100l洗脱缓冲液EB。室温放置10min,12,000rpm离心2min,收集质粒溶液到离心管中。
(3)酶切鉴定,酶切反应体系如表3所示;
表3:酶切反应体系
37℃,反应10分钟。取酶切产物5ul,加入6×loading Buffer 1ul,混匀,加样于1.5%琼脂糖凝胶中,室温下恒压120V电泳30分钟,鉴定酶切是否正确。
(4)测序:将酶切正确的质粒送测序,并与标准序列进行比对,得到最终正确的质粒,由上海英潍捷基公司完成。测序正向引物为5’-CTCCCCTTCAGCTGGACAC-3’,反向引物为5’-AGCTGGGCCACGATTGAC-3’。不同碱基对应的TALEN DNA序列及氨基酸序列如表4所:
表4:同碱基对应的TALEN DNA序列及氨基酸序列
4、体外转录TALEN质的左、右臂mRNA
(1)体外转录模板的制备
1)线性化TALEN质粒(限制性内切酶Not I),以作为体外转录模板。酶切体系如表5所示:
表5:体外转录模板反应体系
37℃,反应2小时。取3μl进行1.5%琼脂糖凝胶电泳,以明确质粒是否酶切完全。
2)酶切产物纯化:北京天根生物有限公司;
使用普通纯化产物试剂盒,纯化回收酶切后产物,详细步骤如下:向吸附柱CB2中加入500ul平衡液BL,12,000rpm离心1min,倒掉收集管中的废液,将吸附柱CB2放回收集管中。向酶切反应液中,加入5倍体积结合液PB,充分混匀。将所得溶液加入吸附柱CB2中,室温放置10min,12,000rpm离心30-60sec,倒掉收集管中的废液,将吸附柱CB2放入收集管中。向吸附柱CB2中加入600ul漂洗液PW,12,000rpm离心30-60sec,弃废液,将吸附柱CB2放入收集管中。重复洗涤一次。将吸附柱CB2放回收集管中,12,000rpm离心2min,尽量除去漂洗液。将吸附柱CB2于室温中放置数分钟,彻底地晾干。最后将吸附柱CB2放入干净的离心管中,向吸附膜中间悬空滴加30ulDEPC处理水,室温放置5min。12,000rpm离心2min收集溶液。
3)模板质粒浓度的测量
取样品2ul加入98ul的洗脱液TE,用Nano drop分光光度计,在ssDNA模式下检测。检测结果显示OD260/OD280介于1.7-1.9之间,说明纯度较好。
(2)体外转录:Life Techologies公司;
使用mMessagemMachine SP6kit试剂盒,进行mRNA合成反应。反应体系如表6所示:
表6:mRNA合成反应体系
根据质粒浓度调整加入量,保证质粒模板总量1ug,轻轻混匀后,37℃水浴2小时。加1ul TURBO Dnase,混匀37℃水浴15min,以消除DNA模板。
(3)转录产物的纯化(氯化锂沉淀)
使用mMessagemMachine SP6kit试剂盒,进行mRNA纯化及重悬,将试剂:RNA 20μl;Licl 30μl;Nuclease Free water 30μl,混匀后,-20℃沉淀大于30min,13200rpm 4℃离心15min,去上清(注意EP管底部RNA沉淀),1ml 70%乙醇清洗,13200rpm 4℃离心15min,去上清。稍晾干后加入Nuclease Free water重悬RNA,测定浓度后,分装,-80℃保存。
4、注射,鉴定及杂交
(1)将终浓度为400ng/μl的TALEN左、右臂的mRNA混合物注射到10个具有Tg(flk1:Egfp/gata1:dsRed)转基因背景的斑马鱼胚胎中,养至成鱼得到F0代,随后将F0代成鱼与野生型斑马鱼交配,获得杂合子F1代,将F1进行基因组DNA测序,鉴定得到+2bp突变体和-7bp突变体杂合子,其中有6个胚胎出现突变,突变效率为60%。
(2)为了净化转基因遗传背景,分别将+2bp和-7bp两种突变体的杂合子与野生型斑马鱼交配3代,得到F2。
(3)分别将+2bp突变体与+2bp突变体交配,-7bp突变体与-7bp突变体交配,最终获得RNF213基因完整敲除+2bp突变体纯合子和-7bp突变体纯合子。
5、观察表型
通过激光片层显微镜及单光子显微镜观察rnf213a-/-表型,发现突变体(+2bp:49.4%;-7bp:46.4%)在后脑静脉与原始后脑交通交界处存在类似烟雾状的血管芽生,该表型类似烟雾病(如图2A-C);更为重要的是在后脑静脉之间出现了桑葚样血管芽生(+2bp:69.4%;-7bp:71.4%),该表型类似人的海绵状血管瘤(如图2D-F),进一步在海绵状血管瘤病人进行检验并得到证实,证实了RNF213基因是海绵状血管瘤的易感基因。这两个表型均在两种基因型(+2bp及-7bp)得到证实,进一步证实了RNF213基因与表型的特异性。因此该基因型斑马鱼(rnf213a-/-)可作为海绵状血管瘤及烟雾病的斑马鱼动物模型。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
SEQUENCE LISTING
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Claims (9)
1.一种rnf213a基因完整敲除纯合子斑马鱼的制备方法,其特征在于,包括以下步骤:
S1、设计针对斑马鱼rnf213a基因2号外显子靶序列的TALEN序列;
S2、构建TALEN左、右臂质粒,并分别体外转录出TALEN左、右臂mRNA;
S3、将TALEN左、右臂mRNA混合并注射到转基因斑马鱼胚胎中,提取胚胎DNA测序发现TALEN的mRNA能作用于rnf213a基因2号外显子;
S4、将注射后的胚胎培养至F0代,并与野生型成鱼交配,获得F1代成鱼;
S5、鉴定F1是否存在rnf213a基因突变,并经DNA测序鉴定rnf213a基因突变类型,将含有rnf213a基因突变的杂合子与野生型外交3代,净化其遗传背景;
S6、将同种基因型的杂合子杂交得到纯合子两种基因型:+2bp及-7bp。
2.如权利要求1所述的制备方法,其特征在于,所述步骤S1中的斑马鱼rnf213a基因2号外显子靶序列为SEQ ID NO.1所示。
3.如权利要求1所述的制备方法,其特征在于,所述步骤S1中的TALEN序列的左臂序列如SEQ ID NO.2所示;右臂序列如SEQ ID NO.3所示。
4.如权利要求1所述的制备方法,其特征在于,所述步骤S1中的TALEN序列还包括能识别不同碱基的核苷酸序列及其编码的氨基酸序列,其中识别碱基A的核苷酸序列如SEQ IDNO.4所示,其编码的氨基酸序列如SEQ ID NO.5所示;识别碱基T的核苷酸序列如SEQ IDNO.6所示,其编码的氨基酸序列如SEQ ID NO.7所示;识别碱基C的核苷酸序列如SEQ IDNO.8所示,其编码的氨基酸序列如SEQ ID NO.9所示;识别碱基G的核苷酸序列如SEQ IDNO.10所示,其编码的氨基酸序列如SEQ ID NO.11所示。
5.如权利要求1所述的制备方法,其特征在于,所述步骤S2中还包括将构建的质粒转化低拷贝的感受态细胞,将其扩大培养后,提取质粒,经限制性内酶切DNA测序鉴定,确定质粒DNA序列。
6.如权利要求5所述的制备方法,其特征在于,所述针对质粒测序设计的正向引物如SEQ ID NO.12所示;反向引物如SEQ ID NO.13所示。
7.如权利要求1所述的制备方法,其特征在于,所述步骤S5中经鉴定含有rnf213a基因突变的成鱼F1杂合子为+2bp突变体及-7bp突变体。
8.如权利要求1所述的制备方法,其特征在于,所述步骤S6中纯合子分别为+2bp突变体及-7bp突变体。
9.一种如权利要求1所述的制备方法建立的rnf213a基因敲除斑马鱼突变体纯合子模型在作为针对烟雾病或/和海绵状血管瘤的药物筛选模型中的用途。
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