DDX4-EGFP transgenic rat model for the study of germline development and spermatogenesis.
Spermatogonial stem cells (SSC) are essential for spermatogenesis and male fertility. In addition, these adult tissue stem cells can be used as vehicles for germline modification in animal models and may have application for treating male infertility. To facilitate the investigation of SSCs and germ lineage development in rats, we generated a DEAD-box helicase 4 (DDX4) (VASA) promoter-enhanced green fluorescent protein (EGFP) reporter transgenic rat. Quantitative real-time polymerase chain reaction and immunofluorescence confirmed that EGFP was expressed in the germ cells of the ovaries and testes and was absent in somatic cells and tissues. Germ cell transplantation demonstrated that the EGFP-positive germ cell population from DDX4-EGFP rat testes contained SSCs capable of establishing spermatogenesis in experimentally infertile mouse recipient testes. EGFP-positive germ cells could be easily isolated by fluorescence-activated cells sorting, while simultaneously removing testicular somatic cells from DDX4-EGFP rat pup testes. The EGFP-positive fraction provided an optimal cell suspension to establish rat SSC cultures that maintained long-term expression of zinc finger and BTB domain containing 16 (ZBTB16) and spalt-like transcription factor 4 (SALL4), two markers of mouse SSCs that are conserved in rats. The novel DDX4-EGFP germ cell reporter rat described here combined with previously described GCS-EGFP rats, rat SSC culture and gene editing tools will improve the utility of the rat model for studying stem cells and germ lineage development.
Department of Obstetrics, Gynecology and Reproductive Sciences and Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, USA.
Kathrin
K
Gassei
Department of Obstetrics, Gynecology and Reproductive Sciences and Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, USA.
Yi
Y
Sheng
Adetunji
A
Fayomi
Sawai Man Singh Medical College and Hospital, Jaipur, India.
Payal
P
Mital
Department of Obstetrics, Gynecology and Reproductive Sciences and Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, USA.
Meena
M
Sukhwani
School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China.
Chih-Cheng
CC
Lin
Biology Department, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
Karen A
KA
Peters
Heart and Vascular Institute, University of Pittsburgh Medical Center, Pittsburgh, PA, USA.
Andrew
A
Althouse
Department of Molecular Genetics and Developmental Biology Graduate Program, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.|Magee-Womens Research Institute, Pittsburgh, Pennsylvania, USA.
Hanna
H
Valli
Department of Obstetrics, Gynecology and Reproductive Sciences and Magee-Womens Research Institute, University of Pittsburgh School of Medicine, Pittsburgh, USA.
Kyle E
KE
Orwig
D000072956:Adult Germline Stem Cells; D000818:Animals; D002478:Cells, Cultured; D053487:DEAD-box RNA Helicases / Q000235:genetics*; D005260:Female; D017930:Genes, Reporter; D005854:Germ Cells / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D008297:Male; D023421:Models, Animal; D011401:Promoter Regions, Genetic; D017207:Rats, Sprague-Dawley; D055647:Rats, Transgenic; D013091:Spermatogenesis
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C091974:Ddx4 protein, rat; D053487:DEAD-box RNA Helicases
DDX4; VASA; germline; rats; spermatogenesis; spermatogonial stem cells; transgenesis