IngentaConnect Bypassing antibiotic selection: positive screening of genetically...

Authors: Kawahara M.¹; Ueda H.^{1, 2}; Morita S.³; Tsumoto K.⁴; Kumagai I.⁴; Nagamune T.¹

Source: Nucleic Acids Research, Volume 31, Number 7, 1 April 2003 , pp. e32-e32(1)

Abstract:

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.

Document Type: Original article

Affiliations: 1: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, 2: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Building FSB-401 Kashiwanoha, Kashiwa 277-8562, Japan, 3: Department of Hematopoietic Factors, The Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan and 4: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan

ArticleBypassing antibiotic selection: positive screening of genetically modified cells with an antigen-dependent proliferation switch

Article
Bypassing antibiotic selection: positive screening of genetically modified cells with an antigen-dependent proliferation switch