Gene Expression in the Preimplantation Bovine Embryo

这项研究将主要在捷克共和国的研究所进行。 《动物生理学和遗传学》是美国国立卫生研究院授予的R37 ND 22681的延伸。 哺乳动物的卵母细胞是一系列有希望的 辅助生殖(人和动物)、保存 遗传多样性，以及通过干细胞技术进行细胞治疗。一个 对成熟卵母细胞的需求增加可以通过丰富的卵子来源来保证 屠宰场卵巢提供的卵母细胞。然而，目前的体外程序， 都不能令人满意，这一点从 导致体外成熟的卵母细胞。对于卵母细胞来说尤其如此。 起源于无数的小有腔卵泡。我们的第一个具体目标是 该项目旨在通过以下方式提高这些卵母细胞的发育能力 建立一种基于两步培养程序的新方法。在……里面 这种方法可逆地抑制卵母细胞的成熟 在特定抑制剂丁内酯I(BLI)的存在下培养它们 一组CDK蛋白。经过长时间的培养后，卵母细胞 在体外成熟和受精；延长的培养期似乎有助于 获得的发展能力会大大减少，如果 卵母细胞在采集后立即成熟。第二个具体问题 AIM将确定RNA干扰，RNAi，以评估基因的适用性 在两步培养过程中衍生的牛胚胎的功能。这个 第二个特定目标将决定RNA干扰，RNAi， 两步法培养牛胚胎基因功能的研究 程序。在这些实验中，双链RNA(RNAi)将被注射到 牛1-细胞胚胎及其促进细胞选择性降解的能力 靶向mRNAs(Polo样激酶，P55 CDC，P55 CDC 后期促进复合体和Securin，它们的破坏需要 姐妹染色单体分离)将通过RT-PCR进行评估。此外，研究还包括 将进行，以确定浓度-和时间的依赖关系 效果，以及定义所需的最小RNAi长度 促进靶向信使核糖核酸降解。

This research will be done primarily in the Czech Republic at the Institute of Animal Physiology and Genetics as an extension of NIH grant R37 ND 22681. The mammalian oocyte is the cornerstone for a wide range of promising technologies such as assisted reproduction (human and animal), preservation of genetic diversity, and cell therapies through stem cell technologies. An increasing demand for mature oocytes could be secured by an abundant source of oocytes provided by abattoir ovaries. The current in vitro procedures, however, are not satisfactory as evidenced by the poor developmental potential of the resulting in vitro matured oocytes. This is especially true for the oocytes derived from the numerous small antral follicles. The first Specific Aim of our project is to enhance the developmental competence of these oocytes by establishing a new method that is based on a two-step culture procedure. In this method the oocytes are reversibly inhibited from undergoing maturation by culturing them in the presence of butyrolactone I (BL I), a specific inhibitor of cdk kinases. Following an extended period of culture the oocytes are then matured and fertilized in vitro; the extended culture period appears to foster the acquisition of developmental competence that is dramatically reduced if the oocytes are matured immediately following their collection. The second Specific Aim will determine the suitability of RNA interference, RNAi, to assess gene function in the bovine embryo derived from the two-step culture procedure. The second Specific Aim will determine the suitability of RNA interference, RNAi, to assess gene function in the bovine embryo derived from the two-step culture procedure. In these experiments double-strand RNA (RNAi), will be injected into 1-cell bovine embryos and its ability to promote the selective degradation of the targeted mRNAs (polo-like kinase, p55 Cdc, a critical subunit of the anaphase-promoting complex, and securin, whose destruction is required for sister chromatid separation) will be assessed by RT-PCR. In addition, studies will be conducted to determine the concentration- and time-dependence of this effect, as well as defining the minimum length of RNAi that is required to promote the degradation of the targeted mRNA.