Assessment of piwil2 gene expression pattern upon germ cell development from mouse embryonic stem cell

ABSTRACT

In order to improve culture conditions for optimal spermatogenesis, quantitative assessment of the male germ cell gene expression profile upon spontaneous ES cell differentiation is necessary. In this study, the quantitative expression profile of Piwil2, germ-line specific marker, during the early stage of embryoid body [EB] formation and differentiation [0-3-day-old EB] was studied. CCE mouse embryonic stem cells [ESCs] were cultured in DMED containing 20% fetal bovine serum [FBS] for 1, 2 and 3 days. The total RNA was isolated from ESCs, 1-3-day-old EBs, and adult testis as positive control. cDNA was prepared and quantitative real-time PCR was done for Oct-4 to study the pluripotency of this cell line. Also, the molecular pattern of PiwiI2 expression in developing EB was investigated. Our quantitative results confirmed the pluripotency of CCE mouse ESC line and showed that PiwiI2 was expressed in undifferentiated CCE mouse ESC line. Our results also showed that expression of Piwil2 increased significantly during the process of EB formation and differentiation up to 2-day-old EB and decreased non- significantly in 3-day-old EB. Our results suggest that EB provide a cellular environment similar to the early embryonic microenvironment and cause the efficient and progressive germ cell lineage differentiation in this system