TY - JOUR
T1 - Tetracycline-regulated gene expression in the NSC-34-tTA cell line for investigation of motor neuron diseases
AU - Babetto, Elisabetta
AU - Mangolini, Alessandra
AU - Rizzardini, Milena
AU - Lupi, Monica
AU - Conforti, Laura
AU - Rusmini, Paola
AU - Poletti, Angelo
AU - Cantoni, Lavinia
PY - 2005/10/31
Y1 - 2005/10/31
N2 - 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.
AB - 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.
KW - Alpha-tubulin
KW - GAP-43
KW - Green fluorescent protein
KW - Neurodegeneration
KW - Neurofilament
KW - NSC-34
KW - Tet-Off
UR - http://www.scopus.com/inward/record.url?scp=26844512590&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=26844512590&partnerID=8YFLogxK
U2 - 10.1016/j.molbrainres.2005.07.010
DO - 10.1016/j.molbrainres.2005.07.010
M3 - Article
C2 - 16125275
AN - SCOPUS:26844512590
SN - 0169-328X
VL - 140
SP - 63
EP - 72
JO - Molecular Brain Research
JF - Molecular Brain Research
IS - 1-2
ER -