MOLECULAR STUDIES OF NK CELLS AND NK/LAK FUNCTION

这项建议的目的是鉴定相应的cDNA克隆 在CD3-NK细胞和其他细胞中优先表达的基因 来表达NK功能，并理解NK的功能 相应的基因产物。一种富含消减文库的方法 对于与NK细胞和NK/LAK活性相关的cDNA克隆将 从克隆的NK细胞(CD3-，CD16+，Leu19+)中制备 表达高水平的NK/LAK功能。各种筛选 方法将在该文库中鉴定感兴趣的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，我们对识别编码细胞的克隆感兴趣 NK细胞和T细胞都表达表面分子；这可能是 确定在调节NK功能中重要的分子。 第三，我们对编码其他分子的克隆感兴趣 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cDNAs 编码膜结合成分或分泌产物的克隆。 首先，用衍生的探针对消减文库进行筛选 从克隆的NK细胞膜结合多聚体中提取Poly-A+RNA 手机。第二，针对NK细胞克隆制备的抗血清和 用LCL吸附后用来探测消减的cDNA文库 在基于lambda的表达载体中制备。长期目标 这个项目的目的是使用从 进一步了解NK细胞生物学的分子技术 细胞，尤其是NK/LAK活性的产生和表达。 而所有要研究的基因都将来自NK克隆 如上所述，探索这些基因在其他 细胞，例如具有NK或NK和LAK功能的CD3+细胞可能会有所帮助 了解这些功能的遗传基础。新定义 将对基因进行测序，并研究它们在不同品种中的表达 关于细胞类型；抗血清/单抗将被制成感兴趣的蛋白质。 虽然难度更大，但我们也计划进行反义(反义 信使核糖核酸)抑制和转染研究。我们预料到 这些研究将提供有用的信息，如 活化的NK细胞被用于临床方案。

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.