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作为NIH资助R37 ND 22681的扩展。 哺乳动物卵母细胞是一系列有前途的 技术，如辅助生殖（人类和动物）， 基因多样性和通过干细胞技术进行的细胞疗法。一个 对成熟卵母细胞的需求不断增加， 由屠宰场卵巢提供的卵母细胞。然而，目前的体外程序， 发展潜力不高， 产生体外成熟的卵母细胞。这对于卵母细胞来说尤其如此 来源于许多小的有腔卵泡。我们的第一个具体目标 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 该方法通过以下方式可逆地抑制卵母细胞经历成熟： 在丁内酯I（BL I）（一种特异性抑制剂）存在下培养它们 CDK激酶。在延长的培养期后， 成熟和体外受精;延长培养期似乎促进 获得发展能力，这是显着减少，如果 卵母细胞在收集后立即成熟。第二特定 目的是确定RNA干扰技术是否适用于评估基因 在牛胚胎中的功能来自两步培养程序。的 第二个具体目标将确定RNA干扰，RNAi， 评估两步培养牛胚胎中的基因功能 procedure.在这些实验中，将双链RNA（RNAi）注射到 1-细胞牛胚胎及其促进选择性降解的能力 靶向的mRNA（polo样激酶，p55 Cdc， 后期促进复合物，和securin，其破坏是必需的， 姐妹染色单体分离）将通过RT-PCR评估。另外研究 将进行，以确定浓度和时间的依赖性，这 影响，以及定义所需的RNAi的最小长度， 促进靶向mRNA的降解。

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.