title: Tetracycline-regulated gene expression in the NSC-34-tTA cell line for investigation of motor neuron diseases.
abstract: The motor neuron-like cell line NSC-34 has become a widely used in vitro model for motor neuron biology and pathology. We established a tetracycline-regulated gene expression system in this cell line by stably transfecting pTet-Off, which codifies for the tetracycline transactivator, the regulatory protein tTA. The monoclonal cell lines (NSC-34-tTA) were evaluated for the presence of functional tTA after transient transfection with pBI-EGFP, analyzing the expression of the reporter gene enhanced green fluorescent protein. We evaluated the regulation of tTA function with doxycycline using fluorescence microscopy and quantitative cytofluorimetric analysis on viable transfected cells. The best-regulated cell line (NSC-34-tTA40) had a 66.4-fold induction for the reporter gene fluorescence in comparison to NSC-34. Alpha-tubulin, GAP-43 and phosphorylated medium and heavy neurofilaments, proteins of importance for the motor neuronal phenotype, were evident in NSC-34-tTA40 by Western blot and immunocytochemistry; they were expressed similarly in NSC-34-tTA40 and in NSC-34. The cDNA of human Cu/Zn superoxide dismutase, a gene of interest for amyotrophic lateral sclerosis, was cloned into pBI-EGFP, downstream of the tetracycline-responsive bidirectional promoter. This plasmid was transiently transfected into NSC-34-tTA40, and the functionality of bidirectional transcription was verified by determining the expression of enhanced green fluorescent protein and of human Cu/Zn superoxide dismutase. Both proteins were regulated by doxycycline. This novel cell line, NSC-34 tTA40, that permits tetracycline-regulated gene expression may prove useful to unravel the mechanisms of motor neuron degeneration.
authors.affiliation: Laboratory of Molecular Pathology, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milan, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Babetto
authors.forename: Alessandra
authors.initials: A
authors.lastname: Mangolini
authors.forename: Milena
authors.initials: M
authors.lastname: Rizzardini
authors.forename: Monica
authors.initials: M
authors.lastname: Lupi
authors.forename: Laura
authors.initials: L
authors.lastname: Conforti
authors.forename: Paola
authors.initials: P
authors.lastname: Rusmini
authors.forename: Angelo
authors.initials: A
authors.lastname: Poletti
authors.forename: Lavinia
authors.initials: L
authors.lastname: Cantoni
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D016472:Motor Neuron Disease / Q000235:genetics*; D009046:Motor Neurons; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013482:Superoxide Dismutase / Q000235:genetics; D000072105:Superoxide Dismutase-1; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection
chemical_list: D017931:DNA Primers; C000606290:SOD1 protein, human; D049452:Green Fluorescent Proteins; C000606291:Sod1 protein, mouse; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D013752:Tetracycline