MOLECULAR ANALYSIS OF THE CORTICAL GRANULE ENVELOPE

描述(改编自申请者的描述)：皮质 颗粒被膜(CGE)是受精后形成的一种主要的包膜。 卵黄周空间，在那里它被假设为阻止多精受精和 在真兽类着床前发育中的作用，包括 人类。研究人员的长期目标是确定其构成的特征 和专家咨询小组的职能。CGE至少包含一种蛋白质p75，它 是本文研究的重点。此应用程序的主要目标是 P75基因的分离和测序及制作所需的分子工具 用于后续对p75的功能研究。第一个具体目标是 测定NH2末端的部分氨基酸序列和几个 P75的内源多肽。这些序列数据将是确认 在特定的目标#2中已经分离出正确的基因。 具体目的是分离和测序该基因(cDNA和基因组DNA)，以用于 第75页。小鼠卵巢基因表达文库的筛选 P75抗体用于鉴定含有p75插入片段的克隆。P75 插入片段将被测序，并在DNA和蛋白质水平上与数据进行比较 在Genbank中以及在目标#1中获得的内部和NH2末端数据。 小鼠p75基因将被用来筛选Stratagene 129/Sv小鼠 基因组文库，整个基因序列将被确定。这一步 是以后构建靶向载体以产生 基因敲除老鼠。第三个特定的目标是制造更多的抗体 重组p75。P75基因将在大肠杆菌中表达，以获得 在兔体内产生抗体所需的足够蛋白质。这将产生一个 足够数量的抗体用于后续的功能 实验。在本项目中获得的数据将为本项目提供重要的基础 P75和CGE的信息，将有助于我们了解整个过程 哺乳动物受精的可能性，并可能导致开发更好的方法来 控制哺乳动物的生殖，包括人类。这些目标也将 提供随后确定p75功能所需的工具和 CGE采用基因打靶和抗体抑制实验。

DESCRIPTION (Adapted from the Applicant's Description): The cortical granule envelope (CGE) is a major post-fertilization coat that forms in the perivitelline space, where it has been hypothesized to block polyspermy and function in preimplantation development of eutherian mammals, including the human. The investigator's long term goal is to characterize the composition and function of the CGE. The CGE contains at least one protein, p75, which is the focus of this study. The major goals of this application are to isolate and sequence gene for p75 and to produce the molecular tools needed for subsequent functional studies of p75. The first Specific Aim is to determine the partial amino acid sequences of the NH2 terminus and several internal peptides for p75. These sequence data will be necessary to confirm that the correct gene has been isolated in Specific Aim #2. The second Specific Aim is to isolate and sequence the gene (cDNA and genomic DNA) for p75. A mouse ovarian cDNA expression library will be screened with an antibody to p75 to identify clones containing the p75 insert. The p75 insert will be sequenced and compared at the DNA and protein levels to data in Genbank and to the internal and NH2 terminus data obtained in Aim #1. Mouse p75 cDNA will then be used to screen the Stratagene 129/Sv mouse genomic library, and the entire gene sequence will be determined. The step is necessary for later construction of a targeting vector to produce knockout mice. The third Specific Aim is to make additional antibodies to recombinant p75. The gene for p75 will be expressed in E. Coli to obtain enough protein for antibody production in rabbits. This will yield a sufficient quantity of antibodies to use in subsequent functional experiments. Data obtained in this project will provide important basic information of p75 and the CGE, will help us understand the overall process of mammalian fertilization, and may lead to development of better ways to control reproduction in mammals, including humans. These Aims will also provide the tools needed to subsequently determine the function of p75 and the CGE using gene targeting and antibody inhibition experiments.