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  • A potential use of embryonic stem cell medium for the in vitro culture of preimplantation embryos. 21617931

    PURPOSE: To assess the impact of embryonic stem cell culture medium (ESCM) on the pre- and post-implantation development of the mouse embryo, as a mammalian model, in comparison with the conventional culture medium, a potassium simplex optimized medium (KSOM). METHODS: Development in ESCM versus KSOM was compared in terms of embryo morphology, cleavage, cavitation, hatching, cell number, expression of TE and ICM transcription factors (Cdx2 and Oct4, respectively), implantation, and development in utero. RESULTS: An enriched medium like ESCM can be beneficial for in vitro embryo development when cultured from the 8-cell stage, as evidenced by promotion of blastocyst development with respect to cavity expansion, hatching, and cell division. Such benefits were not observed when embryos were cultured from the 2-cell stage. CONCLUSIONS: ESCM may augment in vitro embryo development from the 8-cell stage. Using different culture media at different stages may be beneficial to achieve more effective human in vitro fertilization.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • In vivo genetic manipulation of the rat trophoblast cell lineage using lentiviral vector delivery. 19444902

    In this report, we have adapted a lentiviral gene delivery technique for genetic modification of the rat trophoblast cell lineage. Blastocysts were incubated with lentiviral particles and transferred into the uteri of pseudopregnant female rats, harvested at various times during gestation, and then analyzed. Two test systems were evaluated: (1) delivery of an enhanced green fluorescent protein (EGFP) gene under the control of constitutive promoters to rat blastocysts; (2) delivery of EGFP short hairpin RNA (shRNA) to rat blastocysts constitutively expressing EGFP. Lentiviral packaged gene constructs were efficiently and specifically delivered to all trophoblast cell lineages. Additionally, lentiviral mediated transfer of shRNAs was an effective strategy for modifying gene expression in trophoblast cell lineages. This technique will permit the in vivo evaluation of "gain-of-function" and "loss-of-function" manipulations in the rat trophoblast cell lineage.
    Document Type:
    Reference
    Product Catalog Number:
    AB3080
    Product Catalog Name:
    Anti-Green Fluorescent Protein Antibody

  • The Nucleolus in the Mouse Oocyte is Required for the Early Step of Both Female and Male Pronucleus Organization. 20519829

    During oocyte growth in the ovary, the nucleolus is mainly responsible for ribosome biogenesis. However, in the fully-grown oocyte, all transcription ceases, including ribosomal RNA synthesis, and the nucleolus adopts a specific monotonous fibrillar morphology without chromatin. The function of this inactive nucleolus in oocytes and embryos is still unknown. We previously reported that the embryo lacking an inactive nucleolus failed to develop past the first few cleavages, indicating the requirement of a nucleolus for preimplantation development. Here, we reinjected the nucleolus into oocytes and zygotes without nucleoli at various time points to examine the timing of the nucleolus requirement during meiosis and early embryonic development. When we put the nucleolus back into oocytes lacking a nucleolus at the germinal vesicle (GV) stage and at second metaphase (MII), these oocytes were fertilized, formed pronuclei with nucleoli and developed to full term. When the nucleolus was reinjected at the pronucleus (PN) stage, most of the reconstructed zygotes cleaved and formed nuclei with nucleoli at the 2-cell stage, but the rate of blastocyst formation and the numbers of surviving pups were profoundly reduced. Moreover, the zygotes without nucleoli showed a disorder of higher chromatin organization not only in the female pronucleus but also, interestingly, in the male pronucleus. Thus, the critical time point when the nucleolus is required for progression of early embryonic development appears to be at the point of the early step of pronucleus organization.
    Document Type:
    Reference
    Product Catalog Number:
    07-452
    Product Catalog Name:
    Anti-dimethyl-Histone H3 (Lys27) Antibody

  • Cross-regulation of the Nanog and Cdx2 promoters. 19564890

    The first cell fate choice in the mammalian embryo, the segregation of the inner cell mass (ICM) and trophectoderm (TE), is regulated by the mutually antagonistic effects of the transcription factors, Oct4 and Cdx2, while the pluripotency factor, Nanog, is essential to specify the epiblast. We have analyzed the promoters of Nanog and Cdx2, and have found that these two transcription factors are likewise regulated reciprocally. Using an embryonic stem cell line with conditional TE differentiation, we show that Nanog overexpression suppresses the upregulation of TE markers, while Nanog knockdown upregulates the expression of TE markers. We further show that Nanog and Cdx2 bind to and repress each other's promoters. However, whereas Nanog knockout results in detectable Cdx2 expression in the ICM, we observe no overt disruption of blastocyst development, indicating that Nanog plays a subservient role to Oct4 in segregation of the ICM and TE.
    Document Type:
    Reference
    Product Catalog Number:
    05-516
    Product Catalog Name:
    Anti-Ras Antibody, clone RAS10
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