Generation of a Cre-LoxP mouse line expressing hCXCR1

表达 hCXCR1 的 Cre-LoxP 小鼠系的生成

• 批准号: 7048767
• 负责人: MANUELA M. MARTINS-GREEN
• 金额: $ 7.49万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-02-15 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7048767
• 关键词: chemokine; genetically modified animals; human; inflammation; laboratory mouse; receptor; recombinase; role; tissues

DESCRIPTION (provided by applicant): The overall objective of this project is to generate a transgenic mouse line that expresses the human CXCR1 gene in a Cre-recombinase-driven tissue-specific manner as a tool to study CXCR1-dependent chemokine functions. Chemokines are important molecules playing many roles in physiological and pathological conditions, lnterteukine-8 is one of the chemokines that has been intensively studied because it functions in inflammation, wound healing, angiogenesis and tumorigenesis. Much work has been performed to elucidate the functions of this chemokine but progress has been impaired by the lack of an animal model system that can be genetically manipulated. In humans, IL-8 interacts with two receptors, CXCR1 and CXCR2, hence in order to determine how IL-8 functions it is necessary to study the role of each of these receptors in response to activation by hlL-8. These receptors and r|L-8 are highly homologous to the human proteins and rabbits express both receptors. However, it is very difficult (if at all possible) and expensive to perform gene manipulations in rabbits. Mice, on the other hand, do not have the CXCR1 gene or IL-8, hence cannot be used to fully study the functions of IL-8 in humans. We propose here a plan to create a human CXCR1 transgenic mouse line that can serve as an in vivo experimental system to elucidate the functions of IL-8. The specific aims of the work are: 1) To build and test the DMA construct for the transgenic animal. 2) To produce the founder transgenic mice. 3) To test the transgenic mouse line for tissue-specific Cre- recombinase activation. We will first generate a LoxP-based conditional transgenic founder line in which hCXCRI expression is suppressed in all tissues of the mouse. To do so, a DNA transgenic construct will be prepared by molecular cloning methods, tested in a cell culture system to confirm its functionality, including the transgene hCXCRI expression, and Cre-recombinase mediated recombination. We will then supply the DNA transgenic construct to Xenogen Biological that will create the founder transgenic mice. Finally, we will acquire specific Cre-recombinase expressing mice and cross them with our founder mice to achieve hCXCRI transgene activation in the skin to test the functionality of the mouse transgenic line in vivo. The mice expressing hCXCRI in specific tissues will serve as powerful tools to perform research related to human interieukin-8 because delivered hlL-8 will be able to interact with hCXCRI and mCXCR2 (already shown to occur) in the pertinent tissues, providing insight into the molecular mechanisms of the functions of IL-8 in humans. In general, the transgenic animal we are proposing to build will be a useful tool that brings versatility to our research and those of others for understanding the functions of hlL-8.

描述（由申请人提供）：本项目的总体目标是产生以Cre重组酶驱动的组织特异性方式表达人CXCR 1基因的转基因小鼠系，作为研究CXCR 1依赖性趋化因子功能的工具。趋化因子是一种重要的分子，在生理和病理过程中起着重要的作用，其中白细胞介素8（Interteukine-8）在炎症、创伤愈合、血管生成和肿瘤发生中起着重要作用，是目前研究最多的趋化因子之一。已经进行了大量的工作来阐明这种趋化因子的功能，但由于缺乏可以遗传操纵的动物模型系统，进展受到了阻碍。在人中，IL-8与两种受体CXCR 1和CXCR 2相互作用，因此为了确定IL-8如何起作用，有必要研究这些受体中的每一种响应于hIL-8的活化的作用。这些受体和受体|L-8与人蛋白高度同源，兔表达这两种受体。然而，在兔子中进行基因操作是非常困难的（如果可能的话）和昂贵的。另一方面，小鼠没有CXCR 1基因或IL-8，因此不能用于充分研究IL-8在人体中的功能。我们在这里提出了一个计划，建立一个人CXCR 1转基因小鼠系，可以作为一个在体内的实验系统，以阐明IL-8的功能。本工作的具体目的是：1）构建和检测转基因动物的DNA构建体。2)以制造出创始人转基因小鼠。3)测试转基因小鼠系的组织特异性Cre重组酶激活。我们将首先产生基于LoxP的条件性转基因建立系，其中hCXCRI表达在小鼠的所有组织中被抑制。为此，将通过分子克隆方法制备DNA转基因构建体，在细胞培养系统中进行测试以确认其功能性，包括转基因hCXCRI表达和Cre重组酶介导的重组。然后，我们将向Xenogen Biological提供DNA转基因构建体，以创建创始人转基因小鼠。最后，我们将获得特异性Cre重组酶表达小鼠，并将其与我们的创始小鼠杂交，以实现皮肤中的hCXCRI转基因激活，从而在体内测试小鼠转基因系的功能。在特定组织中表达hCXCRI的小鼠将用作进行与人白细胞介素-8相关的研究的有力工具，因为递送的hIL-8将能够与相关组织中的hCXCRI和mCXCR 2（已经显示发生）相互作用，从而提供对人中IL-8功能的分子机制的了解。总的来说，我们提出构建的转基因动物将是一个有用的工具，为我们的研究和其他人的研究带来多功能性，以了解hIL-8的功能。