AMEGA: antigen-mediated genetically modified cell amplification.
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.
Kawahara
Masahiro
M
Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Bunkyo, Tokyo 113-8656, Japan. kawahara@bio.t.u-tokyo.ac.jp
Ueda
Hiroshi
H
Tsumoto
Kouhei
K
Kumagai
Izumi
I
Nagamune
Teruyuki
T
D000941:Antigens / Q000737:chemistry* / Q000235:genetics; D002460:Cell Line; D049452:Green Fluorescent Proteins; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D007202:Indicators and Reagents / Q000737:chemistry; D007377:Interleukin-3 / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017467:Receptors, Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D014162:Transfection / Q000379:methods*
D000941:Antigens; D007135:Immunoglobulin Variable Region; D007202:Indicators and Reagents; D007377:Interleukin-3; D008164:Luminescent Proteins; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C421864:hen egg lysozyme; D009113:Muramidase