title: Production of offspring from a germline stem cell line derived from prepubertal ovaries of germline reporter mice.
abstract: We investigated whether DEAD-box polypeptide 4 (DDX4) positive cells from post-natal ovaries of germline lineage reporter mice can be isolated based on endogenously expressed fluorescent proteins and used to establish a cell line for producing offspring.
  
  
  
  DDX4-positive cells from post-natal ovaries of germline lineage reporter mice can be isolated and used to establish a cell line for producing offspring.
  
  
  
  In recent years, female germline stem cells (FGSCs) have been isolated from the ovaries of post-natal mice by magnetic-activated cell sorting or fluorescence-activated cell sorting (FACS) relying on an antibody against DDX4. However, whether DDX4-positive cells from post-natal ovaries of germline lineage reporter mice can be established without using an antibody, as well as a cell line established for producing offspring, remains unknown.
  
  
  
  To obtain the expected offspring (Ddx4-Cre;mT/mG mice), Ddx4-Cre mice were crossed with mT/mG mice. In the ovaries of Ddx4-Cre;mT/mG mice, germ cells were destined to express enhanced green fluorescent protein (EGFP) while somatic cells still express tandem dimer Tomato (tdTomato). Therefore, the germ cells could be clearly distinguished from somatic cells by fluorescent proteins. Then, we investigated the pattern of fluorescent cells in the ovaries of 21-day-old Ddx4-Cre;mT/mG mice under a fluorescent microscope. Germ cells were sorted by FACS without using antibody and used to establish a FGSC line. The FGSC line was analyzed by DDX4 immunostaining, Edu (5-ethynyl-2'-deoxyuridine) labeling, and RT-PCR for germ cell markers. Finally, the physiological function of the FGSC line was examined by transplanting FGSCs into the ovaries of sterilized recipients and subsequent mating.
  
  
  
  Firstly, we have successfully isolated FGSCs from the ovaries of 21-day-old Ddx4-Cre;mT/mG mice based on endogenously expressed fluorescent proteins. FACS was used to separate the cells and 2.3% of all viable cells was EGFP-positive germ cells. Subsequently, a FGSC line was established that was doubly positive for DDX4 immunostaining and Edu labeling. The mRNA expression of several germ cell markers in this cell line, such as Ddx4, Deleted in azoospermia-like (Dazl), B lymphocyte-induced maturation protein-1 (Blimp1), Stella and Fragilis, was detected. Lastly, the FGSC line was proven to be functional under physiological conditions, as offspring were produced after transplanting FGSCs into ovaries of sterilized recipients and a subsequent mating.
  
  
  
  The molecular mechanisms of proliferation and differentiation of FGSCs in vivo and in vitro still need to be elucidated.
  
  
  
  Our results confirm that DDX4-positive cells can be separated from post-natal mouse ovaries and used to establish cell lines that are functional in producing offspring, and provide further evidence for the existence of post-natal FGSCs in mammals. The Ddx4-Cre;mT/mG mouse strain is an ideal model for the isolation, characterization and propagation of FGSCs and is a useful tool for fully elucidating the molecular mechanisms of proliferation and differentiation of FGSCs in vivo and in vitro.
  
  
  
  none.
  
  
  
  This work was supported by National Basic Research Program of China (2013CB967401) and the National Nature Science Foundation of China (81370675, 81200472 and 81421061). The authors declare no competing interests.
authors.affiliation: Renji Hospital Shanghai Jiaotong University School of Medicine, Key Laboratory for the Genetics of Developmental & Neuropsychiatric Disorders (Ministry of Education), Bio-X Institutes, Shanghai Jiao Tong University, Shanghai 200240, China.
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Renji Hospital Shanghai Jiaotong University School of Medicine, Key Laboratory for the Genetics of Developmental & Neuropsychiatric Disorders (Ministry of Education), Bio-X Institutes, Shanghai Jiao Tong University, Shanghai 200240, China Key Laboratory of Fertility Preservation and Maintenance of Ministry of Education, Ningxia Medical University, Yinchuan 750004, China Shanghai Key Laboratory of Reproductive Medicine, Shanghai 200025, China jiwu@sjtu.edu.cn.
authors.forename: Ji
authors.initials: J
authors.lastname: Wu
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation / Q000502:physiology; D002478:Cells, Cultured; D053487:DEAD-box RNA Helicases / Q000378:metabolism; D005260:Female; D005854:Germ Cells / Q000166:cytology / Q000378:metabolism; D007150:Immunohistochemistry; D051379:Mice; D010053:Ovary / Q000166:cytology* / Q000378:metabolism*; D011627:Puberty / Q000502:physiology
chemical_list: C090864:Ddx4 protein, mouse; D053487:DEAD-box RNA Helicases
keywords: Cre; Ddx4; female germline; reporter mice; stem cells; transplantation