Consequently, nuclear levels of Brg1 were substantially lower in embryos that had arrested at the two-cell stage following interphase enucleation; the fluorescence intensity of immunolabeled Brg1 was reduced to less than 10% of control (Fig. reached by the first mitosis. Our findings demonstrate the amazing flexibility of the reprogramming process and support the importance of nuclear transcriptional regulators in mediating reprogramming. (C Mouse Genome Informatics) (Bultman et al., 2006). We therefore considered the possibility that interphase enculeation was depleting the early embryo of Brg1, which is a required component of the Swi/SNF chromatin remodeling complex and is essential for normal ZGA. We found Levcromakalim that Brg1 could be readily detected by immunostaining in the maternal and paternal pronuclei of interphase zygotes, as well as in the nuclei of two-cell-stage embryos (Fig. 4A,B). However, when interphase zygotes were enucleated, the majority of Brg1 was removed from the cell (Fig. 4C). Consequently, nuclear levels of Brg1 were substantially lower in embryos that had arrested at the two-cell stage following interphase enucleation; the fluorescence intensity of immunolabeled Brg1 was reduced to less than 10% of control (Fig. 4D,H). Although we did observe some residual Brg1 that originated from either RNA or cytoplasmic protein pools, the vast majority of this protein was removed by interphase enucleation. Thus, the failure of interphase nuclear transfer embryos to undergo normal ZGA could be the result of depletion of the Brg1 protein and/or other transcriptional regulators. Open in a separate windows Fig. 4. Brg1 is usually associated with chromatin in interphase zygotes and is excluded in mitosis. (A-G) Levcromakalim Localization of Brg1 in control and in nuclear transfer embryos. (A) A zygote in interphase. (B) Two-cell-stage unmanipulated control embryo. (C) Zygote nucleus removed from an interphase zygote. (D) Two-cell-stage embryo after enucleation in interphase and transfer of a mitotic ES cell genome. One of the two cells is usually shown at high magnification (note size bars). (E) Metaphase zygote arrested with nocodazole. (F) Zygote without genome and removed nuclear material (arrow) of a metaphase zygote arrested in nocodazole. (G) Two-cell-stage embryo after genome removal in mitosis and transfer of a mitotic ES cell genome. (H) Relative quantification of Brg1 nuclear staining. Interphase and mitosis indicate the time point of enucleation. Error bars represent the s.d. of at least three different cells. A hallmark of mitotic entry is usually breakdown of the nuclear envelope and dispersion of many nuclear factors throughout the cytoplasm, which allows the two resulting daughter cells to inherit equal portions of nuclear components. When the localization of Brg1 was assessed in mitotic zygotes, we found that it too was scattered throughout the cytoplasm and excluded from the chromatin (Fig. 4E,F). The cell-cycle dependence of Brg1 localization we observed was consistent with that previously reported in somatic cells and in mouse oocytes, in which Brg1 localizes to the interphase nucleus, but is usually dispersed in the cytoplasm during mitosis (Muchardt et al., 1996; Sun et al., 2007). As a result, when recipient cell chromosome extraction was performed after mitotic entry, Brg1 was not depleted and the resulting two-cell embryos Levcromakalim (Fig. 4G) had Brg1 levels comparable to those of the control two-cell embryos (Fig. 4B,H) and developed normally. Thus the removal of Brg1, and likely many other transcription factors, with the interphase nucleus correlated with developmental failure, whereas the retention of these factors correlated with normal development and successful transcriptional reprogramming. Factors required for reprogramming associate closely with chromatin in interphase but not in mitosis We next considered whether or not performing interphase enucleation via a method that would allow the zygote to maintain a subset of its nuclear factors would stimulate its capacity to develop after nuclear transfer. Recently, a novel method for interphase enucleation has been developed. Instead Levcromakalim of aspirating the entire nucleus from the zygote, the nucleus is usually mechanically disrupted, and the nuclear envelope with attached chromatin is usually more specifically removed (see Fig. S3A in the supplementary material) (Greda et al., 2006). This disruption of the nuclear envelope might be expected to release some nuclear components into the cytoplasm, allowing them to be left behind after removal of the chromatin. We removed the chromosomes from interphase zygotes either by conventional enucleation or by mechanically disrupting the nucleus prior to removing the chromatin. We then transferred nuclei or mitotic chromosomes from various donor cell types into these recipients TIMP3 and compared the extent and efficiency of development (Fig. 5A-F). As had been previously reported (McGrath and Solter, 1984), when eight-cell-stage donor nuclei were injected into normally enucleated.