Dub-2−/− embryos fail to hatch in vitro.

A) Genotyping by genomic PCR. 330 bp, amplified by using Dub F and Dub R primers, represents either Dub-2+/+ or Dub-2+/−, whereas no band represents Dub-2−/−. Dub-2−/− embryos were found between 0.5dpc and 3.5dpc. B) Out-growth inner cell mass was harvested and then genotype was confirmed by 2-primers PCR (Dub F and Dub R) as described in Materials and Methods. Developmental defects in Dub-2−/− embryos at the 5.5dpc stage were shown in vitro. ICM grew normally and was surrounded by trophoblast giant cells in Dub-2+/+ and Dub-2+/− embryos. In contrast, Dub-2−/−5.5dpc embryos did not hatch to the culture dish. C) The number of hatching status of 3.5dpc embryos after 96 hrs of culture in vitro. Dub-2 deficiency leads to failure to hatch in vitro. D) Analysis of TUNEL assay was performed for cell death. Arrows pointing spots indicate cells undergoing apoptosis. E) E3.5 blastocysts were isolated, and after 24 hrs in culture, their ZP was mechanically removed. The ZP-free embryos were cultured on feeder layer in ES cell medium. F)Dub-2+/+ and Dub-2+/− mES cell lines.