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）：该项目的总体目标是产生一种转基因小鼠系，该小鼠系以 Cre 重组酶驱动的组织特异性方式表达人类 CXCR1 基因，作为研究 CXCR1 依赖性趋化因子功能的工具。趋化因子是在生理和病理条件下发挥多种作用的重要分子，Interteukine-8是已被深入研究的趋化因子之一，因为它在炎症、伤口愈合、血管生成和肿瘤发生中发挥作用。人们已经开展了大量工作来阐明这种趋化因子的功能，但由于缺乏可进行基因操纵的动物模型系统，进展受到了损害。在人类中，IL-8 与两种受体 CXCR1 和 CXCR2 相互作用，因此为了确定 IL-8 如何发挥作用，有必要研究这些受体中的每一种在响应 hIL-8 激活时的作用。这些受体和 r|L-8 与人类蛋白高度同源，兔子表达这两种受体。然而，在兔子身上进行基因操作非常困难（如果可能的话）并且昂贵。另一方面，小鼠没有CXCR1基因或IL-8，因此不能用于充分研究IL-8在人类中的功能。我们在此提出了创建人类 CXCR1 转基因小鼠系的计划，该小鼠系可以作为体内实验系统来阐明 IL-8 的功能。这项工作的具体目标是： 1) 构建并测试转基因动物的 DMA 结构。 2) 产生创始转基因小鼠。 3) 测试转基因小鼠系的组织特异性Cre重组酶激活。我们将首先生成基于 LoxP 的条件转基因创始系，其中 hCXCRI 表达在小鼠的所有组织中均受到抑制。为此，将通过分子克隆方法制备 DNA 转基因构建体，并在细胞培养系统中进行测试以确认其功能，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，我们将获得表达特定 Cre 重组酶的小鼠，并将它们与我们的创始小鼠杂交，以在皮肤中实现 hCXCRI 转基因激活，以测试小鼠转基因系的体内功能。在特定组织中表达 hCXCRI 的小鼠将成为进行与人 interieukin-8 相关的研究的强大工具，因为递送的 hIL-8 将能够与相关组织中的 hCXCRI 和 mCXCR2（已显示发生）相互作用，从而提供对相关组织的深入了解。 IL-8 在人类中的功能的分子机制。总的来说，我们打算构建的转基因动物将是一个有用的工具，为我们和其他人的研究带来多功能性，以了解 hIL-8 的功能。