Novel approaches for disrupting gene expression in mammalian oocytes

破坏哺乳动物卵母细胞基因表达的新方法

基本信息

批准号：
8195724
负责人：
JANICE P EVANS
金额：
$ 24.6万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-08-24 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8195724
关键词：
Ablation Affinity Area Binding Biological Biology Cell Cycle Regulation Cell Nucleus Cells Clinical Trials Complex Contraceptive methods Cultured Cells Deoxyribonucleotides Development Disease Double-Stranded RNA Drug Delivery Systems Female Female Contraceptive Agents Fertility Fertilization Foundations Future Gene Delivery Gene Expression Gene Targeting Genetic Transcription Goals Human In Vitro Infertility Investigation Isomerism Knock-out Knockout Mice Knowledge Libraries Malignant neoplasm of prostate Mediating Meiotic Prophase I Messenger RNA Methodology Methods Microinjections Modification Mus Nucleic Acids Oligonucleotides Oocytes PLK1 gene Peptides Phenotype Process Proteins RNA RNA Interference Reagent Reproductive Health Research Research Methodology Research Proposals Residual state Small Interfering RNA Surface Testing Therapeutic Time Transcript Transfection Transgenic Organisms Vesicle Virus Diseases Work ZP1 ZP2 Zona Pellucida aptamer base cancer cell cell type ds-DNA genetic manipulation human disease in vitro testing in vivo insight interest mRNA Transcript Degradation novel novel strategies protein degradation tool zona pellucida glycoprotein

项目摘要

DESCRIPTION (provided by applicant): This R21 project seeks to develop new approaches for the genetic manipulation of mammalian oocytes, which we envision will accelerate advancing our knowledge of oocyte function and reproductive health. Genetic manipulation of mammalian oocytes has primarily used two methods: RNA interference and knockout mice. While RNAi has been a highly successful method of RNA ablation and subsequent protein knockdown in oocytes, RNAi approaches are not without limitations, as knockdown can be inefficient. Furthermore, double-stranded RNA and siRNAs have to be introduced into oocytes by microinjection, which is labor- and time-intensive and makes it impractical do use a large-scale RNAi approach (e.g., siRNA library-based screens). Knockout mice certainly have provided significant insights into mammalian oocyte biology as well, but knockout approaches also are not without pitfalls, including the time and expense involved in obtaining a knockout. This project seeks to develop alternatives to these methods, utilizing different established nucleic acid-based methods in novel combinations and with specialized modifications for the applications proposed here. In Aim 1, we will augment the use of siRNAs for post-transcriptional gene silencing with another reagent, a short single-stranded nucleic acid called a triplex-forming oligonucleotide (TFO) for pre-transcriptional silencing. TFOs bind to homopurine tracts in double-stranded DNA, and have been used to regulate gene expression in cultured cells and in vivo. The hypothesis for Aim 1 is that TFOs will inhibit transcription of a targeted gene, while siRNAs will mediate degradation of any residual mRNAs that were transcribed. This will be tested in vitro with isolated oocytes as well as with follicle-enclosed oocytes for longer-term culture. In Aim 2, we will develop methods for delivery of agents into oocytes. We will identify a novel agent for oocyte- specific delivery, using a screen of an aptamer library (with 1.2 X 1018 oligo-2'-deoxyribonucleotide sequence isomers) to isolate an aptamer that will interact with the oocyte's zona pellucida (ZP). Aptamers are nucleic acid-based molecules that bind with high affinity to target molecules. Aptamers can be used for delivery of agents such as siRNAs into cells; this delivery works in vivo, and aptamers currently are being developed as therapeutics to target drugs and other agents to specific cell types for treatment of a variety of diseases (13 aptamers are in clinical trials). Additionally, as an alternative tool, we will also test a cell-penetrating peptide for intra-oocyte delivery. We will couple siRNAs or TFOs to ZP-binding aptamers and/or a cell-penetrating peptide, and test these for their actions in oocytes. The future direction of this work will be to test the ZP-targeting aptamer for systemic delivery of siRNAs and TFOs, as a means of in vivo oocyte-specific knockdown as an alternative to knockout/transgenic methodologies, as well as potentially the foundation of a novel female contraceptive. PUBLIC HEALTH RELEVANCE: Investigations of the biology of the mammalian oocyte are of significant value, both for increasing our basic biological knowledge and for applications to human reproductive health. This project will accelerate the pace of research in this important area of reproductive health by developing new research methods for use in studying oocyte biology and female fertility and infertility, and for development of new female contraceptive methods.

描述（由申请人提供）：该 R21 项目旨在开发哺乳动物卵母细胞基因操作的新方法，我们预计这将加速推进我们对卵母细胞功能和生殖健康的了解。哺乳动物卵母细胞的基因操作主要使用两种方法：RNA干扰和基因敲除小鼠。虽然 RNAi 是一种非常成功的 RNA 消融和随后卵母细胞蛋白质敲低的方法，但 RNAi 方法并非没有局限性，因为敲低可能效率低下。此外，双链RNA和siRNA必须通过显微注射引入卵母细胞，这是劳动密集型和时间密集型的，并且使得使用大规模RNAi方法（例如基于siRNA库的筛选）变得不切实际。敲除小鼠当然也为哺乳动物卵母细胞生物学提供了重要的见解，但敲除方法也并非没有缺陷，包括获得敲除所需的时间和费用。该项目旨在开发这些方法的替代方案，以新颖的组合利用不同的已建立的基于核酸的方法，并针对此处提出的应用进行专门的修改。在目标 1 中，我们将使用另一种试剂（一种称为三链体形成寡核苷酸 (TFO) 的短单链核酸，用于转录前沉默）来增强 siRNA 在转录后基因沉默中的使用。 TFO 与双链 DNA 中的同型嘌呤束结合，并已用于调节培养细胞和体内的基因表达。目标 1 的假设是 TFO 将抑制目标基因的转录，而 siRNA 将介导转录的任何残留 mRNA 的降解。这将用分离的卵母细胞以及用于长期培养的卵泡封闭的卵母细胞进行体外测试。在目标 2 中，我们将开发将药剂递送至卵母细胞的方法。我们将通过筛选适体库（具有 1.2 X 1018 寡-2'-脱氧核糖核苷酸序列异构体）来分离与卵母细胞透明带 (ZP) 相互作用的适体，从而确定一种用于卵母细胞特异性递送的新型试剂。适体是基于核酸的分子，能够以高亲和力与靶分子结合。适体可用于将 siRNA 等试剂递送至细胞中；这种传递在体内发挥作用，目前正在开发适配体作为治疗剂，将药物和其他药剂靶向特定细胞类型，以治疗多种疾病（13 种适配体正在临床试验中）。此外，作为替代工具，我们还将测试用于卵母细胞内递送的细胞穿透肽。我们将 siRNA 或 TFO 与 ZP 结合适体和/或细胞穿透肽偶联，并测试它们在卵母细胞中的作用。这项工作的未来方向将是测试用于 siRNA 和 TFO 系统递送的 ZP 靶向适体，作为体内卵母细胞特异性敲除的一种手段，作为敲除/转基因方法的替代方法，并可能为新型女性避孕药。公共健康相关性：哺乳动物卵母细胞的生物学研究对于增加我们的基础生物学知识和在人类生殖健康中的应用具有重要价值。该项目将通过开发用于研究卵母细胞生物学、女性生育力和不孕症的新研究方法以及开发新的女性避孕方法，加快生殖健康这一重要领域的研究步伐。