Expressed genes in interleukin-4 treated B cells identified by cDNA representational difference analysis
Expressed genes in interleukin-4 treated B cells identified by cDNA representational difference analysis
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DOI：
10.1016/s0161-5890(98)00031-5
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发表时间：
1998-06-01
影响因子：
3.6
通讯作者：
Paul, WE
中科院分区：
文献类型：
--
作者：
Chu, CC;Paul, WE
白介素 4 (IL-4) 刺激 B 细胞生长和分化，例如诱导成熟 B 细胞转而产生 IgG1 和 IgE。为了进一步表征 IL-4 对 B 细胞的影响，我们使用灵敏的基于 PCR 的消减方法来分离 IL-4 处理细胞中表达的基因。我们的方法结合了基因组代表性差异分析 (RDA) 方法对 cDNA 分析的适应性与物理分离方法（磁珠去除）。这种 cDNA RDA 技术使我们能够对相对少量的高度、特征化、纯化的 B 细胞进行扣除，并且可以方便地制备。在受刺激的 B 细胞中，来自 LPS + IL-4 刺激的 B 细胞的 cDNA。两轮消减导致预期 IL-4 诱导的 C gamma 1 cDNA 增强超过 100 倍。此时，我们克隆了这个消减文库，并分析了 154 个随机挑选的克隆的序列相似性。从这些克隆中获得了 37 个单独的基因。大多数这些基因 (30) 可以通过序列相似性进行功能鉴定。这些包括编码 C gamma 1 (1)、细胞骨架成分 (4) 和参与 DNA 复制的产物 (3) 的基因。代谢（5）、信号转导（4）、转录（4）、翻译（6）和运输（3）。只有 7 个基因与 GenBank、EMBL 或 Swiss Prot 数据库中的已知序列没有相似性。 Northern印迹分析证实，一个未知基因（IL-4诱导基因1的图1）和一个与人转录因子E4BP4同源的基因被IL-4处理诱导了10-20倍。 LPS + IL-4 处理的 B 细胞中表达的基因列表可能有助于进一步了解 IL-4 刺激细胞的作用和机制。 (C) 1998 Elsevier Science Ltd. 保留所有权利。
Interleukin-4 (IL-4) stimulates B cell growth and differentiation, such as inducing mature B cells to switch to IgG1 and IgE production. To further characterize IL-4 effects on B cells, we used a sensitive PCR-based subtraction approach to isolate genes expressed in IL-4 treated cells. Our approach combined an adaptation of the genomic representational difference analysis (RDA) method to cDNA analysis with a physical separation method (magnetic bead depletion). This cDNA RDA technique allowed us to perform subtraction on the relatively small number of highly, characterized, purified B cells that can be conveniently prepared. In stimulated B cells from cDNA from LPS + IL-4 stimulated B cells. Two rounds of subtraction resulted in greater than 100-fold enhancement of expected IL-4-induced C gamma 1 cDNA. At that point, we cloned this subtraction library and analysed 154 randomly picked clones for sequence similarities. From these clones, 37 individual genes were obtained. Most of these genes (30) could be functionally identified by sequence similarity. These included genes encoding C gamma 1 (1), cytoskeletal components (4) and products involved in DNA replication (3). metabolism (5), signal transduction (4), transcription (4), translation (6) and transport (3). Only 7 genes had no similarity to known sequences in the GenBank, EMBL or Swiss Prot databases. One unknown gene (designated Fig1 for IL-Four Induced Gene 1) and one gene with homology to the human transcription factor E4BP4 were confirmed by Northern blot analysis to be induced 10-20-fold by IL-4 treatment. This list of expressed genes in LPS + IL-4 treated B cells may shed further insight on the action and mechanism of IL-4 stimulation of cells. (C) 1998 Elsevier Science Ltd. All rights reserved.