Bypassing antibiotic selection: positive screening of genetically modified cells with an antigen-dependent proliferation switch
Bypassing antibiotic selection: positive screening of genetically modified cells with an antigen-dependent proliferation switch
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DOI：
10.1093/nar/gng032
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发表时间：
2003-04-01
影响因子：
14.9
通讯作者：
Nagamune, T
中科院分区：
文献类型：
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作者：
Kawahara, M;Ueda, H;Nagamune, T
虽然抗生素选择已常规用于选择遗传修饰的细胞，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。在这项研究中，我们提出了一个抗原介导的遗传修饰细胞扩增（AMEGA）系统，采用抗体/受体嵌合体没有抗生素的选择。基于其中二聚体促红细胞生成素受体（EpoR）或gp 130的胞外结构域被抗鸡蛋溶菌酶（HEL）抗体的异二聚体V-H/V-L区和EpoR D2结构域取代的合理设计，编码嵌合体以及模型转基因增强型绿色荧光蛋白（EGFP）的基因，逆转录病毒感染到IL-3依赖性Ba/F3细胞中，然后在不存在IL-3的情况下直接进行HEL选择。在单轮选择后，选择性扩增EGFP阳性细胞，导致几乎100%阳性细胞的群体。没有抗生素选择的AMEGA不会伤害正常细胞，这将特别有助于提高基于干细胞的基因治疗的功效。
While antibiotic selection has been routinely used for the selection of genetically modified cells, administration of cytotoxic drugs often leads to deleterious effects not only to inert cells but also to transfected or transduced ones. In this study, we propose an Antigen-MEdiated Genetically modified cell Amplification (AMEGA) system employing antibody/receptor chimeras without antibiotic selection. Based on a rational design where the extracellular domains of dimeric erythropoietin receptor (EpoR) or gp130 were substituted with heterodimeric V-H/V-L regions of anti-hen egg lysozyme (HEL) antibody and EpoR D2 domains, the genes encoding the chimeras as well as a model transgene, enhanced green fluorescent protein (EGFP), were retrovirally infected into IL-3-dependent Ba/F3 cells followed by direct HEL selection in the absence of IL-3. After a single round of selection, EGFP-positive cells were selectively amplified, resulting in a population of almost 100% positive cells. The AMEGA without antibiotic selection will not harm normal cells, which will be especially useful for increasing the efficacy for stem cell-based gene therapy.