Cellular commitment to mortality: analyses of mouse early embryo

细胞对死亡的承诺：小鼠早期胚胎的分析

• 批准号: 7963998
• 负责人: Minoru Ko
• 金额: $ 28.24万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7963998
• 关键词: Aging; Cells; Classification; DNA; DNA Microarray Chip; Development; Embryo; Fertilization; Gene Activation; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Genomics; Goals; In Situ Hybridization; Molecular; Molecular Profiling; Morula; Mus; Names; Organ; Pattern; Play; Proteins; RNA; RNA Degradation; Reverse Transcriptase Polymerase Chain Reaction; Role; Source; Staging; Techniques; Therapeutic; Tissues; base; blastocyst; embryonic stem cell; falls; gene function; implantation; mortality; nuclear reprogramming; preimplantation; promoter; stem cell biology

We carried out DNA microarray-based global expression profiling of all preimplantation stages in mouse. It revealed and characterized the distinctive patterns of maternal RNA degradation and two major transient waves of de novo transcription: zygotic genome activation (ZGA) and mid-preimplantation gene activation (MGA). We also developed a technique to do large-scale Whole Mount In Situ Hybridization (WISH), which allows us to reveal the spatial expression patterns of 91 genes in mouse preimplantation embryos. Through these analyses, we identified a number of genes that show unique expression patterns. We have demonstrated that one of them, named Zscan4, is expressed exclusively in 2-cell mouse embryos and ES cells and plays a critical role in the progression of preimplantation development. Another gene (Chuk) shows constant RNA levels throughout preimplantation development and can be used as an internal standard suitable for quantitative RT-PCR. We also identified genes, named Trim43a, Trim43b, and Trim43c, whose expression began at the 2-cell stage, peaked at the 8-cell/morula stage, and dramatically fell by the blastocyst stage. We identified a 5 kb DNA fragment that covers upstream region of Trim43a as a putative promoter, which can drive the expression of mStrawberry fluorescent protein in a manner similar to endogenous Trim43 genes. Trim43 genes will be useful stage-specific markers for the study of preimplantation embryos. We are currently studying the functions of these genes in detail.

我们进行了基于DNA微阵列的全球表达谱的所有植入前阶段的小鼠。它揭示和特点的独特模式的母体RNA降解和两个主要的瞬时波从头转录：合子基因组激活（ZGA）和中期植入前基因激活（MGA）。我们还开发了一种技术，做大规模的整体原位杂交（WISH），这使我们能够揭示小鼠植入前胚胎中91个基因的空间表达模式。通过这些分析，我们确定了一些显示独特表达模式的基因。我们已经证明，其中一个，命名为Zscan 4，只在2-细胞小鼠胚胎和ES细胞中表达，并在植入前发育的进展中起着关键作用。另一个基因（Chuk）在整个着床前发育过程中显示出恒定的RNA水平，可以用作定量RT-PCR的内标。我们还鉴定了名为Trim 43 a、Trim 43 b和Trim 43 c的基因，其表达开始于2细胞期，在8细胞/桑椹胚期达到峰值，并在囊胚期急剧下降。我们鉴定了一个覆盖Trim 43 a上游区域的5 kb DNA片段作为推定的启动子，它可以以类似于内源Trim 43基因的方式驱动mStrawberry荧光蛋白的表达。Trim 43基因将成为研究植入前胚胎发育阶段特异性的有用标记。我们目前正在详细研究这些基因的功能。