TRANSGENIC STUDIES OF LEUKOCYTE CD11A PROMOTER

白细胞CD11A启动子的转基因研究

基本信息

批准号：
2734911
负责人：
KINDRED A RITCHIE
金额：
$ 8.37万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-07-01 至 2000-06-30
项目状态：
已结题

项目摘要

This proposal aims to use the leukocyte integrin CD11a (leukocyte function antigen-1, LFA-1) promoter to direct tissue and stage specific expression of reporter genes in transgenic mice. The integrin gene family codes for heterodimers which mediate cell to cell and cell to matrix adherence. CDI Ia belongs to the beta-2 subclass, or leukocyte, integrins, which mediate cellular adherence in inflammation and the immune response. Leukocyte integrins are responsible for leukocyte margination and emigration into inflamed tissues, and function as co-receptors in tumor and microbial killing and T-cell activation. The leukocyte integrin molecules consist of one of three alpha chains (CD11a, CD11b, or CD11c) noncovalently bound to a single common beta chain (CD18) to form three different heterodimers. Although CD18 is expressed on all leukocytes, CD11a is expressed on T and B lymphocytes, monocytes, and macrophages. The leukocyte integrins are also differentially expressed in leukocyte ontogeny, showing, in general, increased expression with leukocyte maturation, differentiation, and activation. In vitro transfection studies of CD11a-promoter -human growth hormone reporter gene constructs have defined regions of the CD11a promoter responsible for high level, tissue specific expression.The experiments described in this proposal will investigate the regulation of the CD11a promoter in transgenic mice. A construct consisting of 0.8 kilobases of the human CD11a promoter driving a human CD4-human growth hormone minigene is currently being injected into mouse oocytes. The cytoplasmic portion of the CD4 gene has been deleted to reduce the possibility of intracellular signalling via associated protein kinases so that CD4 may serve only as a cell surface reporter molecule. Initial experiments will detect the integrated transgene and characterize its expression. Lineage specific expression on leukocytes will be assayed by FACS analysis of peripheral blood and lymph node leukocytes . Tissue specific expression will be further characterized by screening various tissues for transgene mRNA using RNA blots and a reverse transcriptase-PCR assay. Additional regulatory sequences will be delineated by injecting other constructs having additional 5', intronic, and 3' sequences from the human CD11a gene . This approach, plus a comparison with parallel transgenic studies using the human CD11b and CD18 promoters should delineate regulatory regions responsible for differential gene expression in lymphocytes and monocytes/macrophages and provide insight into the sequences responsible for coordinate regulation of the CD11 and CD18 subunits during hematopoietic cell differentiation. Long-term plans involve using the CD11a promoter to: 1) express inducible cellular toxins in leukocytes to create an animal model in which to study the role of leukocytes in inflammation; and 2) express selected oncogenes in leukocytes to study the differentiation of B cells and monocytes/macrophages. In sum, these experiments will investigate the regulation of a)11a, an important leukocyte adherence molecule, and use the CD11a promoter in transgenic mice to study leukocyte function and ontogeny.

该建议旨在使用白细胞整合蛋白CD11A（白细胞功能抗原1，LFA-1）启动子直接组织和分期特定表达转基因小鼠中的报告基因。整合素基因家族代码异二聚体，将细胞介导细胞和细胞的基质粘附。 CDI ia属于介导的Beta-2子类或白细胞的整合素炎症和免疫反应中的细胞粘附。白细胞整联蛋白负责白细胞边缘和移民到发炎的组织，并充当肿瘤和微生物的共受体杀死和T细胞激活。白细胞整联蛋白分子由三个α链（CD11A，CD11B或CD11C）中的三个无共价结合单个常见的β链（CD18）形成三种不同的异二聚体。尽管CD18在所有白细胞上表达，但CD11a在T和 B淋巴细胞，单核细胞和巨噬细胞。白细胞结构是在白细胞个体发育中也差异表达，通常显示通过白细胞成熟，分化和激活。 CD11A促进剂 - 人类生长的体外转染研究激素报告基因构建体已定义了CD11a的区域负责高水平，组织特异性表达的启动子。本提案中描述的实验将调查调节转基因小鼠中的CD11a启动子。由0.8组成的构造人类CD11a启动子的千目标驱动人类CD4-Human生长当前，荷尔蒙微型基因被注射到小鼠卵母细胞中。这 CD4基因的细胞质部分已被删除以减少通过相关蛋白激酶的细胞内信号传导的可能性该CD4只能用作细胞表面报告基因分子。最初的实验将检测到集成的转基因并表征其表达。白细胞上的谱系特定表达将由外周血和淋巴结白细胞的FACS分析。组织特定表达将通过筛选各种表达来进一步表征使用RNA印迹和逆转录酶-PCR的转基因mRNA组织测定。将通过注射来划定其他调节序列其他具有额外5'，内含子和3'序列的构造人CD11a基因。这种方法，以及与平行的比较使用人CD11b和CD18启动子的转基因研究应划定负责差异基因表达的调节区域在淋巴细胞和单核细胞/巨噬细胞中，并洞悉负责坐标CD11和CD18的序列造血细胞分化过程中的亚基。长期计划涉及使用CD11a启动子：1）表达可诱导的细胞毒素在白细胞中创建一个动物模型，以研究其作用炎症中的白细胞； 2）在白细胞研究B细胞和单核细胞/巨噬细胞。总而言之，这些实验将研究调节A）11a，一个重要的白细胞依从性分子，并使用转基因小鼠中的CD11a启动子研究白细胞功能和个体发育。