title: 1-NGFR receptor is a new flow cytometric tool for rapid cell cycle-correlated gene therapy complementation studies in viable cells.
abstract: Control of successful genetic complementation of cellular defects in heterogenous cell populations requires biochemical selection markers or cell analytical specifiers. With available gene therapy technologies, only a fraction of a cell population is transfected or transduced. We applied and optimized a novel dual-laser flow cytometric technique to analyze immediately the genetic complementation of cells dysregulated in cell proliferation and DNA repair. A novel bicistronic retrovirus carrying the normal Fanconi anemia gene (group C) and the cell surface marker gene l-NGFR was constructed to analyze the normalization of the G2-phase cell cycle defect in DNA-repair-deficient FA(C) lymphoblastoid cells after transduction. Using a dual-laser multiparameter technology, we 1) analyzed the cell-cycle distribution of viable/dead cells using Hoechst 33342 (with ultraviolet light), 2) identified the genetically complemented cells by FITC antibody labeling of the novel l-NGFR surface marker (488 nm), and 3) recorded mitomycin C-induced cell death in nontransduced and transduced cells by propidium iodide. Artificial l-NGFR expression is high and similar in ontogenetically and phylogenetically different cell populations. With this novel l-NGFR marker technology, the success of gene therapy of cell-cycle dysregulation in even small subpopulations of cells can be recorded within 48 h. In addition to improved cell analysis, l-NGFR surface marker expression can also be used for rapid generation of pure cell populations by column cell separation technologies.
authors.affiliation: Boehringer Mannheim, Biotechnology Therapeutics Research Center, Penzberg, Germany. andreas_machl@bmg.boehringer-mannheim.com
authors.forename: A W
authors.initials: AW
authors.lastname: Machl
authors.forename: S A
authors.initials: SA
authors.lastname: Planitzer
authors.forename: M
authors.initials: M
authors.lastname: Kubbies
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001562:Benzimidazoles; D002453:Cell Cycle / Q000235:genetics; D016923:Cell Death / Q000187:drug effects; D002461:Cell Line, Transformed / Q000166:cytology / Q000378:metabolism; D002470:Cell Survival / Q000235:genetics; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D005816:Genetic Complementation Test / Q000379:methods*; D015316:Genetic Therapy; D006801:Humans; D008214:Lymphocytes / Q000166:cytology* / Q000378:metabolism; D016685:Mitomycin / Q000494:pharmacology; D011419:Propidium; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism*; D012190:Retroviridae
chemical_list: D000903:Antibiotics, Antineoplastic; D001562:Benzimidazoles; D005456:Fluorescent Dyes; D020800:Receptor, Nerve Growth Factor; D017475:Receptors, Nerve Growth Factor; D011419:Propidium; D016685:Mitomycin; C017807:bisbenzimide ethoxide trihydrochloride