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MOLECULAR STUDIES OF NK CELLS AND NK/LAK FUNCTION

NK 细胞和 NK/LAK 功能的分子研究

基本信息

批准号：
2060837
负责人：
FRITZ H BACH
金额：
$ 31.57万
依托单位：
BETH ISRAEL DEACONESS MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-07-01 至 1994-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2060837
关键词：
T lymphocyte clone cells gene expression genetic library histocompatibility antigens human subject laboratory mouse laboratory rabbit membrane proteins molecular cloning monoclonal antibody natural killer cells nucleic acid probes nucleic acid sequence transfection

项目摘要

The aim of this proposal is to identify cDNA clones corresponding to genes preferentially expressed in CD3- NK cells and other cells that express NK function and to understand the function of the corresponding gene products. A subtracted cDNA library enriched for cDNA clones associated with NK cells and NK/LAK activity will be prepared from a cloned NK cell (CD3-,CD16+,Leu19+) that expresses high-level NK/LAK function. A variety of screening methods will identify cDNA clones of interest within this library. Our first priority is to identify cDNA clones encoding cell surface molecules associated with NK cells and/or NK and LAK function. Second, we are interested in identifying clones encoding cell surface molecules expressed in both NK and T cells; this may identify molecules important in the regulation of NK function. Third, we are interested in clones that encode other molecules expressed preferentially in NK cells and other cells that express NK function. Two types of probes will be used to identify cDNA clones that encode membrane-bound components or secreted products. First, the subtracted library will be screened with a probe derived from poly-A+ RNA from membrane-bound polysomes of the cloned NK cell. Second, antisera prepared against the NK cell clone and absorbed with LCL will be used to probe the subtracted cDNA library prepared in a lambda-based expression vector. A long-term goal of this project is to use information and reagents obtained by molecular techniques to understand further the cell biology of NK cells, especially the generation and expression of NK/LAK activity. Whereas all genes to be studied will be derived from the NK clone described above, probing for expression of these genes in other cells, e.g. CD3+ cells with NK or NK and LAK function may help understand the genetic basis of these functions. Newly-defined genes will be sequenced and their expression studied in a variety of cell types; antisera/moAb will be made to proteins of interest. Although more difficult, we also plan to perform anti-sense (anti- mRNA) inhibition and transfection studies. We anticipate that these studies will provide information that will be useful as activated NK cells are used in clinical protocols.

该建议的目的是鉴定相应的cDNA克隆，在CD 3- NK细胞和其他细胞中优先表达的基因表达NK细胞功能的细胞，相应的基因产物。富集的消减cDNA文库对于与NK细胞和NK/LAK活性相关的cDNA克隆，从克隆的NK细胞（CD 3-、CD 16+、Leu 19+）制备，表达高水平的NK/LAK功能。多种筛选方法将鉴定该文库中感兴趣的cDNA克隆。我们的首要任务是鉴定编码细胞表面的cDNA克隆，与NK细胞和/或NK和LAK功能相关的分子。第二，我们感兴趣的是鉴定编码细胞的克隆，表面分子在NK细胞和T细胞中都表达;这可能鉴定在NK功能调节中重要的分子。第三，我们对编码其他分子的克隆很感兴趣优先在NK细胞和其他表达 NK功能。将使用两种类型的探针来鉴定cDNA 编码膜结合组分或分泌产物的克隆。首先，将用衍生的探针筛选消减文库。来自克隆NK的膜结合多聚核糖体的poly-A+ RNA cell. 第二，制备抗NK细胞克隆的抗血清，用LCL吸附的DNA探针对消减cDNA文库进行检测在基于拟南芥的表达载体中制备。一个长期目标该项目的目的是利用信息和试剂获得的，进一步了解NK细胞生物学的分子技术尤其是NK/LAK活性的产生和表达。然而，所有待研究的基因都将来自NK克隆，如上所述，探测这些基因在其他细胞中的表达，细胞，例如具有NK或NK和LAK功能的CD 3+细胞可能有助于了解这些功能的遗传基础。新定义基因将被测序，它们的表达将在各种的细胞类型;抗血清/单克隆抗体将被用于感兴趣的蛋白质。虽然难度更大，但我们也计划执行反义（反 mRNA）抑制和转染研究。我们预计这些研究将提供有用的信息，活化的NK细胞用于临床方案。