MOLECULAR ANALYSIS OF THE CORTICAL GRANULE ENVELOPE

皮质颗粒包膜的分子分析

基本信息

批准号：
2674083
负责人：
Prudence Talbot
金额：
$ 7.3万
依托单位：
UNIVERSITY OF CALIFORNIA RIVERSIDE
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-09-01 至 2001-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2674083
关键词：
animal genetic material tag antibody egg /ovum gene targeting genetic library genetically modified animals laboratory mouse laboratory rabbit membrane membrane proteins molecular cloning nucleic acid sequence protein sequence recombinant proteins

项目摘要

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.

描述（改编自申请人的描述）：皮质颗粒包膜（CGE）是受精后形成的主要外衣卵周间隙，被假设可以阻止多精受精在真兽类哺乳动物植入前发育中的功能，包括人类。研究人员的长期目标是表征成分和 CGE 的职能。 CGE 至少含有一种蛋白质 p75，是本研究的重点。该应用程序的主要目标是分离和测序 p75 基因并产生所需的分子工具用于 p75 的后续功能研究。第一个具体目标是确定 NH2 末端的部分氨基酸序列和几个 p75 的内部肽。这些序列数据对于确认是必要的正确的基因已在特定目标 #2 中分离出来。第二个具体目标是分离基因（cDNA 和基因组 DNA）并对其进行测序第75页。小鼠卵巢 cDNA 表达文库将通过 p75 抗体以鉴定含有 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 Genbank 中的数据以及目标 #1 中获得的内部和 NH2 末端数据。然后使用小鼠 p75 cDNA 筛选 Stratagene 129/Sv 小鼠基因组文库，并且将确定整个基因序列。步骤对于以后构建靶向载体以产生基因敲除小鼠。第三个具体目标是制造额外的抗体重组p75。 p75基因将在大肠杆菌中表达以获得足够的蛋白质用于兔子体内产生抗体。这将产生一个足够数量的抗体用于后续功能实验。本项目获得的数据将为 p75和CGE的信息，将帮助我们了解整个流程哺乳动物受精的研究，并可能导致开发更好的方法控制哺乳动物（包括人类）的繁殖。这些目标也将提供随后确定 p75 功能所需的工具和 CGE 使用基因靶向和抗体抑制实验。