If PcG proteins are physiologically involved in the neuroendocrine control of female puberty via repression of the Kiss1 gene in the ARC, preventing the pubertal decrease in PcG gene expression that occurs in this hypothalamic region at the onset of puberty would be expected to delay the pubertal process. Because Eed is required for the silencing activity of the PcG complex 32, we chose to overexpress EED in the ARC of immature female rats. We cloned the coding region of rat Eed tagged with a hemagglutinin epitope into a lentivirus vector (LV) that also expresses eGFP (Supplementary Fig. 7a). After confirming the production of the HA-tagged protein by western blot (Supplementary Fig. 7b) we stereotaxically delivered this construct (termed LV-EED) bilaterally into the hypothalamus of 26-day-old female rats, targeting the ARC. Control animals were injected with a construct expressing only eGFP (LV-eGFP). Immunohistofluorescent analysis of the sites of injection using antibodies against eGFP was used to identify the transduced cells (Fig. 6a). Kisspeptin neurons, also identified by immunohistofluorescence, were one of the cell populations transduced by the virus (Fig. 6b,c).
