Generation of a Cre-LoxP mouse line expressing hCXCR1

描述（由申请人提供）：该项目的总体目标是产生一种以cre重组酶驱动的组织特异性方式表达人类CXCR1基因的转基因小鼠系，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过程中发挥多种作用的重要分子，白细胞介素-8是目前研究较多的趋化因子之一，在炎症、伤口愈合、血管生成和肿瘤发生等方面发挥着重要作用。已经进行了大量的工作来阐明这种趋化因子的功能，但由于缺乏可以基因操纵的动物模型系统，进展受到损害。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在响应hlL-8激活时的作用。这些受体和r|l -8与人蛋白高度同源，家兔均表达这两种受体。然而，在兔子身上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，小鼠不具有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。在此，我们提出了一个建立人类CXCR1转基因小鼠系的计划，该系可以作为体内实验系统来阐明IL-8的功能。工作的具体目的是：1)构建和测试转基因动物的DMA结构。2)产生创始人转基因小鼠。3)验证转基因小鼠系对组织特异性Cre-重组酶的激活作用。我们将首先生成一个基于loxp的条件转基因创始系，其中hCXCRI在小鼠所有组织中的表达被抑制。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，包括转基因hCXCRI的表达和Cre-recombinase介导的重组。然后，我们将提供DNA转基因构建物给Xenogen生物公司，该公司将创建转基因小鼠的创始人。最后，我们将获得特异性表达cre -重组酶的小鼠，并将其与我们的创始小鼠杂交，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的小鼠将成为开展人类白细胞介素-8相关研究的有力工具，因为递送的hlL-8将能够与相关组织中的hCXCRI和mCXCR2相互作用（已经显示发生），从而深入了解IL-8在人类中的功能的分子机制。总的来说，我们提出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了解hlL-8的功能带来多功能性。

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.