Bypassing antibiotic selection: positive screening of genetically modified cells with an antigen-dependent proliferation switch.
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.
Kawahara
Masahiro
M
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan. kawahara@bio.t.u-tokyo.ac.jp
Ueda
Hiroshi
H
Morita
Sumiyo
S
Tsumoto
Kouhei
K
Kumagai
Izumi
I
Nagamune
Teruyuki
T
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000906:Antibodies / Q000235:genetics* / Q000276:immunology; D000941:Antigens / Q000276:immunology; D001665:Binding Sites / Q000235:genetics; D002455:Cell Division / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009113:Muramidase / Q000276:immunology* / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D017467:Receptors, Erythropoietin / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods*
D000900:Anti-Bacterial Agents; D000906:Antibodies; D000941:Antigens; D007377:Interleukin-3; D008164:Luminescent Proteins; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C421864:hen egg lysozyme; D009113:Muramidase