An improved expression vector to create transgenic mice for prion research.

一种改进的表达载体，用于创建用于朊病毒研究的转基因小鼠。

• 批准号: 7208718
• 负责人: GULTEKIN TAMGUNEY
• 金额: $ 7.69万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-01-19 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7208718
• 关键词: improved; expression; vector; create; transgenic

DESCRIPTION (provided by applicant): Prions are transmissible pathogenic agents responsible for fatal neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle. These disorders are characterized by the conversion of the host-encoded cellular prion protein (PrPc) into an abnormally folded and infectious isoform (PrPSc) that accumulates in the brain and causes disease. Transgenic mice are important tools to study prion diseases. Due to the expression vectors in use, current transgenic mouse models are imperfect and show PrPc-expression profiles dissimilar from wild-type (wt) mice. Our long-term goal is to develop a transgenic mouse model in which the PrPc-expression profile is identical to wt PrPc expression. An authentic mouse model is a prerequisite to the development of accurate protocols that prevent the transmission of infectious prions and attenuate or cure the disease. Our specific hypothesis is that a transgenic expression vector based on a mouse bacterial artificial chromosome (BAG) clone will include all genetic regulatory elements necessary to drive a locus-independent expression of PrPc that is indistinguishable from wt mice. The specific aims are to: 1. Develop a new vector for PrPc expression in transgenic mice. Based on a fully sequenced mouse BAG clone, we will generate an array of truncated vectors that can drive PrPc expression in transgenic animals. We will introduce a cloning site into this vector that will allow replacing the open reading frame (ORF) of the prion protein gene (Prnp) with alternative sequences. 2. Create and identify transgenic mice that have a PrPc-expression profile identical to wt mice. We will use the expression vectors cloned in Specific Aim #1 to create transgenic mice through pronuclear microinjection into oozytes from Prnp-ablated mice. We will create and identify animals that have a PrPc- expression profile that is indistinguishable from wt mice. 3. Characterize the incubation time and PrPSc profile in transgenic mice after infection with prions. Once we have identified a vector that gives rise to transgenic animals that have a wt PrPc-expression profile, we will infect these animals with prions. We will determine the incubation time and the PrPSo profile generated in these mice and validate this new mouse model by comparison to wt mice.

描述（由申请方提供）：朊病毒是导致致命性神经退行性疾病（如人类克雅氏病（CJD）、绵羊瘙痒病和牛海绵状脑病（BSE））的传染性病原体。这些疾病的特征在于宿主编码的细胞朊病毒蛋白（PrPc）转化为异常折叠和感染性同种型（PrPSc），其在脑中积累并引起疾病。转基因小鼠是研究朊病毒疾病的重要工具。由于使用的表达载体，目前的转基因小鼠模型是不完善的，并显示出与野生型（wt）小鼠不同的PrPc表达谱。我们的长期目标是开发一种转基因小鼠模型，其中PrPc表达谱与野生型PrPc表达相同。一个真实的小鼠模型是一个先决条件，以制定准确的协议，防止传染性朊病毒的传播和减轻或治愈疾病。我们的具体假设是，基于小鼠细菌人工染色体（BAG）克隆的转基因表达载体将包括驱动与野生型小鼠难以区分的PrPc基因座非依赖性表达所需的所有遗传调控元件。具体目标是：1. PrPc基因在转基因小鼠中表达载体的构建基于完全测序的小鼠BAG克隆，我们将产生一系列截短的载体，可以驱动PrPc在转基因动物中的表达。我们将在该载体中引入一个克隆位点，该位点将允许用替代序列替换朊病毒蛋白基因（Prnp）的开放阅读框（ORF）。2.创建并鉴定具有与野生型小鼠相同的PrPc表达谱的转基因小鼠。我们将使用在特异性目标#1中克隆的表达载体，通过将原核显微注射到来自Prnp消融小鼠的卵中来创建转基因小鼠。我们将创建并鉴定具有与野生型小鼠难以区分的PrPc表达谱的动物。3.表征感染朊病毒后转基因小鼠中的孵育时间和PrPSc谱。一旦我们鉴定出产生具有野生型PrPc表达谱的转基因动物的载体，我们将用朊病毒感染这些动物。我们将确定这些小鼠中产生的孵育时间和PrPSo谱，并通过与野生型小鼠比较来验证这种新的小鼠模型。