Characterization of female germ-like cells derived from mouse embryonic stem cells through expression of GFP under the control of Figla promoter
Characterization of female germ-like cells derived from mouse embryonic stem cells through expression of GFP under the control of Figla promoter
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通过 Figla 启动子控制下的 GFP 表达来表征源自小鼠胚胎干细胞的雌性生殖样细胞
DOI：
10.1002/jcb.24044
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发表时间：
2012-04-01
影响因子：
4
通讯作者：
Hua, Jinlian
中科院分区：
文献类型：
--
作者：
Hu, Yue;Sun, Junwei;Hua, Jinlian
先前的研究表明，生殖细胞可以从小鼠胚胎干细胞（ESCs）中获得。然而，仍然没有有效的系统可以在体外可视化生殖细胞特化的阶段，并进一步鉴定和富集胚胎干细胞来源的生殖细胞。Figla（生殖系中的因子，α）基因编码生殖细胞特异性转录因子，其协调卵母细胞特异性透明卵母细胞（Zp）基因的表达，并且对于小鼠中的卵泡发生是必需的。在此，我们首先构建了在Figla启动子控制下表达增强型绿色荧光蛋白（EGFP）的pFigla-EGFP重组质粒，并产生和表征了稳定携带该pFigla-EGFP报告构建体的ESC系。通过维甲酸（RA）诱导液培养贴壁胚状体（EBs）或将胚胎干细胞与卵巢细胞一起移植到肾被膜下，诱导胚胎干细胞向雌性生殖样细胞分化。一群分化的ESC表达GFP，并通过RT-PCR和免疫荧光分析这些细胞。GFP阳性细胞表达生殖细胞标志物Vasa、减数分裂特异基因Stra8、Scp3、卵母细胞标志物Gdf9、Zp3和Figla，表明该方法可用于雌性生殖细胞的纯化和筛选。我们的研究建立了一种新的雌性生殖样细胞衍生选择体系，为进一步研究干细胞衍生生殖细胞的发育和分化提供了方法。J.细胞。113：11111121，2012中。(c)2011 Wiley Periodicals，Inc.
Previous studies have demonstrated that germ cells can be derived from mouse embryonic stem cells (ESCs). However, there is still no efficient system, which can visualize the stage of germ cell specification in vitro, and further to identify and enrich germ cells derived from ESCs. Figla (factor in the germline, alpha) gene encodes a germ cell specific transcription factor that coordinates the expression of the oocyte-specific zona pellucida (Zp) genes and is essential for folliculogenesis in mouse. Here, we first constructed a pFigla-EGFP recombinant plasmid that expressed enhanced green fluorescent protein (EGFP) under the control of Figla promoter, and generated and characterized an ESC line stably carrying this pFigla-EGFP reporter construct. Then the ESCs were induced to differentiate into female germ-like cells by culturing adherent embryoid bodies (EBs) in retinoic acid (RA) induction medium or transplanting ESCs under the kidney capsule with ovarian cells. A population of differentiated ESCs expressed GFP, and these cells were analyzed by RT-PCR and immunofluorescence. The GFP positive cells showed the expression of germ cell markers Vasa, meiotic specific gene Stra8, Scp3, oocyte markers Gdf9, Zp3 and Figla, indicating that this method could be used for the purification and selection of female germ cells. Our study establishes a new selective system of female germ-like cell derivation and offers an approach for further research on the development and the differentiation of germ cells derived from stem cells. J. Cell. Biochem. 113: 11111121, 2012. (c) 2011 Wiley Periodicals, Inc.