公开(公告)号：US6127176A

公开(公告)日：2000-10-03

申请号：US95485

申请日：1998-06-10

申请人： George R. Stark ,  Xiaoxia Li

发明人： George R. Stark ,  Xiaoxia Li

IPC分类号： C07K14/715 ,  C12N5/22 ,  C12N5/10 ,  C12N5/16

CPC分类号： C07K14/7155

摘要： Mutant cell lines which have lost their ability to respond to IL-1 are provided. In one embodiment, the mutant cell line lacks or is essentially free of IL-1 receptor associate kinase (IRAK), an IL-1 signaling pathway component. The present invention also provides a method for making mutant cell lines that are unresponsive to IL-1, TNF, or to both cytokines. The method comprises the steps of: transfecting cells with a Herpes Simplex Virus thymidine kinase (HSV-TK) gene and a second gene for positive selection, each of said genes being operatively linked to an IL-1 inducible promoter or a TNF inducible promoter; selecting for transfected cells that express HSV thymidine kinase and the product of the positive selection gene in response to IL-1 or TNF; determining a gancyclovir concentration which kills the selected cells in the presence of IL-1 or TNF and which does not kill the selected cells in the absence of IL-1 or TNF; mutagenizing the selected cells using a chemical mutagenizing agent; and treating the mutagenized cells with the determined concentration of gancyclovir and IL-1 or TNF. The present invention also relates to a method of identifying domains or amino acids in IRAK that are essential for IRAK to function in the IL-1 signaling pathway.

摘要翻译： 提供了失去其对IL-1反应能力的突变细胞系。 在一个实施方案中，突变细胞系缺少或基本上不含IL-1受体缔合激酶（IRAK），IL-1信号传导途径组分。 本发明还提供了制备对IL-1，TNF或两种细胞因子无反应的突变细胞系的方法。 该方法包括以下步骤：用单克隆单克隆病毒胸苷激酶（HSV-TK）基因转染细胞，第二基因用于阳性选择，每个所述基因与IL-1诱导型启动子或TNF诱导型启动子有效连接; 选择表达HSV胸苷激酶的转染细胞，以及响应于IL-1或TNF的阳性选择基因的产物; 确定在存在IL-1或TNF的情况下杀死所选择的细胞并且在不存在IL-1或TNF的情况下不杀死所选细胞的gancyclovir浓度; 使用化学诱变剂诱变所选择的细胞; 并用确定浓度的甘氨酸和IL-1或TNF处理诱变的细胞。 本发明还涉及鉴定IRAK中在IL-1信号传导途径中起关键作用的IRAK中的结构域或氨基酸的方法。

公开(公告)号：US4302204A

公开(公告)日：1981-11-24

申请号：US54200

申请日：1979-07-02

申请人： Geoffrey M. Wahl ,  George R. Stark

发明人： Geoffrey M. Wahl ,  George R. Stark

IPC分类号： C12N15/10 ,  C12Q1/68 ,  C12Q1/6832 ,  C12Q1/6834 ,  G01N31/22 ,  G01N33/16 ,  G01N33/48

CPC分类号： C12Q1/6832 ,  C12N15/1034 ,  C12Q1/6834 ,  Y10S435/805

摘要： Improvements in the transfer and detection of separated nucleic acids, both RNA and DNA, are provided. For analysis of large DNA, the molecular weight segregated fractions of DNA are depurinated and fragmented to provide fractions having less than about 2 kb as single strands. With both RNA and DNA, the nucleic acid fractions are transferred after resolution to a chemically treated substrate and covalently affixed to the substrate. The resulting nucleotides affixed to the substrate are hybridized with labeled nucleotide probes and a volume exclusion agent, particularly a water soluble ionic polymer.

摘要翻译： 提供分离的核酸（RNA和DNA）的转移和检测的改进。 为了分析大的DNA，DNA的分子量分离级分被去除并分离，以提供具有小于约2kb的单链的级分。 使用RNA和DNA，将核酸级分在分辨后转移到化学处理的底物上并共价固定在底物上。 附着于底物的所得核苷酸与标记的核苷酸探针和体积排阻剂，特别是水溶性离子聚合物杂交。