l-NGFR receptor is a new flow cytometric tool for rapid cell cycle-correlated gene therapy complementation studies in viable cells

Autor: Simone A Planitzer, Andreas W Machl, Manfred Kubbies
Rok vydání: 1997
Předmět:
Zdroj: Cytometry. 29:371-374
ISSN: 1097-0320
0196-4763
DOI: 10.1002/(sici)1097-0320(19971201)29:4<371::aid-cyto14>3.0.co;2-r
Popis: 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.
Databáze: OpenAIRE
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