GSE122215
Peg10 regulation of TSC differentiation
The prevailing dogma that approximately 50% of our genome is “junk” DNA composed of transposable elements and retroviral insertions has recently been challenged. It has become evident that our genome has taken advantage of these transposable elements and uses them as a source of DNA to generate novel genes, which subsequently allow the organism to evolve. This process is termed “domestication of transposable elements” and the majority of these genes have been found to be essential for the existence of the organism. One of these developmentally essential domesticated genes: Peg10 (paternally expressed gene 10), was derived from a Ty3/gyspy LTR retrotransposon, yet lost its ability to transpose due to mutational events during its domestication. Remarkably, Peg10 has successfully maintained its Gag and Pol-like domains for millions of years. Peg10 orthologues are expressed in eutherian mammals and are essential for placentogenesis. To address the functional mechanisms of Peg10 we studied it in Trophoblast Stem Cells (TSCs). We find that the Gag of Peg10 is fully active: it promotes budding of vesicles, akin to the viral counterpart that catalyzes the budding of viruses. TSCs, deleted for Peg10, fail to differentiate into placental lineages, underscoring a critical role in lineage specification. This paper discusses our efforts to characterize the contents of Peg10 vesicles and whether such vesicles regulate lineage specification. Overall design: RNA was extracted from following genotypes - wildtype TSCs (WT_TSC), Peg10 knockout TSCs (KO_TSC), wildtype TSCs differentiated in 20% oxygen (WT_TSC_diff), Peg10 knockout TSCs differentiated in 20% oxygen (KO_TSC_diff),  wildtype TSCs differentiated in 2% oxygen (WT_diff_2O2),and Peg10 knockout TSCs differentiated in 2% oxygen (KO_diff_2O2). Cells are kept in the pluripotent state by growing them on CellStart/Fgf4/Heparin. The cells were differentiated in two different conditions: 20% oxygen and 2% oxygen. The samples were collected at 10th day following differentiation. Cells are harvested and RNA is isolated using the Qiagen RNeasy kit. RT-PCR was performed for several differentiation markers to validate the success of the assay.