1-NGFR receptor is a new flow cytometric tool for rapid cell cycle-correlated gene therapy complementation studies in viable cells.
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.
Machl
A W
AW
Boehringer Mannheim, Biotechnology Therapeutics Research Center, Penzberg, Germany. andreas_machl@bmg.boehringer-mannheim.com
Planitzer
S A
SA
Kubbies
M
M
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
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