AMEGA: antigen-mediated genetically modified cell amplification
AMEGA: antigen-mediated genetically modified cell amplification
复制标题
DOI：
10.1016/j.jim.2003.10.007
复制
发表时间：
2004-01-01
影响因子：
2.2
通讯作者：
Nagamune, T
中科院分区：
文献类型：
--
作者：
Kawahara, M;Ueda, H;Nagamune, T
选择遗传修饰的细胞是将细胞工程化为具有所需特性的关键步骤。虽然抗生素选择已被普遍使用，但细胞毒性药物的施用通常不仅对惰性细胞而且对转染或转导的细胞产生有害作用。为了克服这个问题，提出了一种用于遗传修饰细胞的阳性筛选方法，其使用一对嵌合受体，所述嵌合受体响应于特定抗原而触发生长信号。将抗鸡蛋溶菌酶（HEL）抗体HyHEL-10的V-H或V-L区与促红细胞生成素受体（EpoR）的胞外D2结构域和EpoR或gp 130的跨膜/胞质结构域融合。将模型转基因、增强型绿色荧光蛋白（EGFP）和重建功能性Fv的嵌合受体基因逆转录病毒共感染白细胞介素（IL）-3依赖性Ba/F3细胞，随后在不存在IL-3的情况下进行直接HEL选择。因此，单轮选择导致单个群体的EGFP阳性细胞。描述了称为抗原介导的遗传修饰细胞扩增（AMEGA）的方法的详细方案。(C)2003 Elsevier B. V.保留所有权利。
Selection of genetically modified cells is a critical step to engineer the cells with desired properties. While antibiotic selection has been commonly used, administration of cytotoxic drugs often leads to deleterious effects not only to inert cells but also to transfected or transduced ones. To overcome this problem, a positive screening method for genetically modified cells is proposed using a pair of chimeric receptors that trigger a growth signal in response to a specific antigen. Either V-H or V-L region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 was fused to extracellular D2 domain of erythropoietin receptor (EpoR) and transmembrane/cytoplasmic domains of either EpoR or gp130. A model transgene, enhanced green fluorescent protein (EGFP) and the chimeric receptor genes that reconstituted functional Fv were retrovirally co-infected to interleukin (IL)-3-dependent Ba/F3 cells, followed by direct HEL selection in the absence of IL-3. Consequently, a single round of selection led to a single population of EGFP-positive cells. The detailed protocol of the method termed antigen-mediated genetically modified cell amplification (AMEGA) is described. (C) 2003 Elsevier B.V. All rights reserved.