本文主要研究内容
作者莫丽芬（2019）在《鸡Dazl荧光报告基因追踪生殖细胞迁移和分化的研究》一文中研究指出：诱导多能干细胞(Induced Pluripotent stem Cell,iPSC)具有分化成多种类型细胞的潜能。其中iPSC在体外诱导分化成生殖细胞对于畜禽保种育种、转基因动物以及发育生物学研究具有重要的意义。Dazl是调控生殖细胞发育与分化的关键因子,是生殖细胞鉴定的主要标志物。由于Dazl在iPSC不表达,因此可以作为报告基因追踪iPSC诱导分化为生殖细胞的过程。本研究利用 CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats,CRISPR)技术介导的同源末端连接(Homology-Mediated End Joining,HMEJ)将红色荧光蛋白基因mCherry定点敲入鸡内源Dazl基因第10个内含子处,通过Dazl启动子驱动mCherry表达,从而精确追踪内源Dazl在iPSC分化为原始生殖细胞(Primordial germ cell,PGC)的过程中的表达情况。具体研究结果如下:(1)DAZL在鸡PGC和iPSC细胞中的表达鉴定。我们首先克隆鸡Dazl基因,制备DAZL多克隆抗体。纯化的抗体对鸡PGC进行Western blot,结果显示DAZL抗体能识别PGC中的特异性条带,相对分子量为33KDa,与预测分子量一致,说明DAZL抗体特异性高。q-PCR和免疫荧光染色结果显示DAZL在iPSC中不表达,在PGC中高表达,说明Dazl能作为报告基因追踪生殖细胞的特异性。(2)Dazl荧光报告载体的构建和定点敲入验证。首先针对Dazl基因设计和构建hpl80-sgRNA-spCas9表达载体和打靶供体质粒pDazl-HMEJ donor。然后将hpl80-sgRNA-spCas9转染DF-1细胞,验证CRISPR/Cas9系统的切割Dazl基因的效率。T7E1酶切鉴定证明hp180-sgRNA-spCas9质粒有效靶向目的位点实现基因敲除。而后将hpl80-sgRNA-spCas9表达载体和pDazl-HMEJ donor报告载体共转染PGC和iPSC,成功建立Dazl-mCherry+/EGFP+ PGC细胞系和Dazl-mCherry/EGFP+ iPSC细胞系。(3)Dazl荧光报告系统示踪PGC细胞迁移和iPSC细胞分化。本实验将Dazl-mCherry+/EGFP+标记的PGC细胞移植到15HH的鸡胚血液系统,结果显示细胞会随着血脉系统迁移并归巢到性腺,并且在新生鸡的睾丸继续发育。说明Dazl荧光报告系统能追踪生殖细胞的迁移。最后,我们将Dazl-mCherry/EGFP+标记的iPSC制作类配体(Embryoid body,EB),并用EB培养液诱导,3天后形成的EB同时表达mCherry和EGFP。将EB收集检测生殖细胞特异性基因的和多能性相关基因的表达,q-PCR结果显示生殖细胞特异性基因显著上调,多能性相关基因显著下调。说明Dazl荧光报告系统在iPSC体外诱导分化过程中可起指示作用。综上所述,本研究利用CRISPR/Cas9技术建立鸡Dazl荧光报告系统,追踪生殖细胞的迁移和体外分化。通过可视化诱导iPSC获得生殖细胞,将为进一步利用iPSC进行珍稀禽类保种育种、转基因鸡制备以及发育生物学研究的奠定基础。
Abstract
you dao duo neng gan xi bao (Induced Pluripotent stem Cell,iPSC)ju you fen hua cheng duo chong lei xing xi bao de qian neng 。ji zhong iPSCzai ti wai you dao fen hua cheng sheng shi xi bao dui yu chu qin bao chong yo chong 、zhuai ji yin dong wu yi ji fa yo sheng wu xue yan jiu ju you chong yao de yi yi 。Dazlshi diao kong sheng shi xi bao fa yo yu fen hua de guan jian yin zi ,shi sheng shi xi bao jian ding de zhu yao biao zhi wu 。you yu Dazlzai iPSCbu biao da ,yin ci ke yi zuo wei bao gao ji yin zhui zong iPSCyou dao fen hua wei sheng shi xi bao de guo cheng 。ben yan jiu li yong CRISPR/Cas9(Clustered regularly interspaced short palindromic repeats,CRISPR)ji shu jie dao de tong yuan mo duan lian jie (Homology-Mediated End Joining,HMEJ)jiang gong se ying guang dan bai ji yin mCherryding dian qiao ru ji nei yuan Dazlji yin di 10ge nei han zi chu ,tong guo Dazlqi dong zi qu dong mCherrybiao da ,cong er jing que zhui zong nei yuan Dazlzai iPSCfen hua wei yuan shi sheng shi xi bao (Primordial germ cell,PGC)de guo cheng zhong de biao da qing kuang 。ju ti yan jiu jie guo ru xia :(1)DAZLzai ji PGChe iPSCxi bao zhong de biao da jian ding 。wo men shou xian ke long ji Dazlji yin ,zhi bei DAZLduo ke long kang ti 。chun hua de kang ti dui ji PGCjin hang Western blot,jie guo xian shi DAZLkang ti neng shi bie PGCzhong de te yi xing tiao dai ,xiang dui fen zi liang wei 33KDa,yu yu ce fen zi liang yi zhi ,shui ming DAZLkang ti te yi xing gao 。q-PCRhe mian yi ying guang ran se jie guo xian shi DAZLzai iPSCzhong bu biao da ,zai PGCzhong gao biao da ,shui ming Dazlneng zuo wei bao gao ji yin zhui zong sheng shi xi bao de te yi xing 。(2)Dazlying guang bao gao zai ti de gou jian he ding dian qiao ru yan zheng 。shou xian zhen dui Dazlji yin she ji he gou jian hpl80-sgRNA-spCas9biao da zai ti he da ba gong ti zhi li pDazl-HMEJ donor。ran hou jiang hpl80-sgRNA-spCas9zhuai ran DF-1xi bao ,yan zheng CRISPR/Cas9ji tong de qie ge Dazlji yin de xiao lv 。T7E1mei qie jian ding zheng ming hp180-sgRNA-spCas9zhi li you xiao ba xiang mu de wei dian shi xian ji yin qiao chu 。er hou jiang hpl80-sgRNA-spCas9biao da zai ti he pDazl-HMEJ donorbao gao zai ti gong zhuai ran PGChe iPSC,cheng gong jian li Dazl-mCherry+/EGFP+ PGCxi bao ji he Dazl-mCherry/EGFP+ iPSCxi bao ji 。(3)Dazlying guang bao gao ji tong shi zong PGCxi bao qian yi he iPSCxi bao fen hua 。ben shi yan jiang Dazl-mCherry+/EGFP+biao ji de PGCxi bao yi zhi dao 15HHde ji pei xie ye ji tong ,jie guo xian shi xi bao hui sui zhao xie mai ji tong qian yi bing gui chao dao xing xian ,bing ju zai xin sheng ji de gao wan ji xu fa yo 。shui ming Dazlying guang bao gao ji tong neng zhui zong sheng shi xi bao de qian yi 。zui hou ,wo men jiang Dazl-mCherry/EGFP+biao ji de iPSCzhi zuo lei pei ti (Embryoid body,EB),bing yong EBpei yang ye you dao ,3tian hou xing cheng de EBtong shi biao da mCherryhe EGFP。jiang EBshou ji jian ce sheng shi xi bao te yi xing ji yin de he duo neng xing xiang guan ji yin de biao da ,q-PCRjie guo xian shi sheng shi xi bao te yi xing ji yin xian zhe shang diao ,duo neng xing xiang guan ji yin xian zhe xia diao 。shui ming Dazlying guang bao gao ji tong zai iPSCti wai you dao fen hua guo cheng zhong ke qi zhi shi zuo yong 。zeng shang suo shu ,ben yan jiu li yong CRISPR/Cas9ji shu jian li ji Dazlying guang bao gao ji tong ,zhui zong sheng shi xi bao de qian yi he ti wai fen hua 。tong guo ke shi hua you dao iPSChuo de sheng shi xi bao ,jiang wei jin yi bu li yong iPSCjin hang zhen xi qin lei bao chong yo chong 、zhuai ji yin ji zhi bei yi ji fa yo sheng wu xue yan jiu de dian ding ji chu 。
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论文作者分别是来自广西大学的莫丽芬，发表于刊物广西大学2019-10-14论文，是一篇关于诱导多能干细胞论文,分化论文,广西大学2019-10-14论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自广西大学2019-10-14论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
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