Characterization of female germ-like cells derived from mouse embryonic stem cells through expression of GFP under the control of Figla promoter

摘要

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.