Proceedings of The Physiological Society
King's College London (2011) Proc Physiol Soc 22, C22
MicroArray analysis of transduced cell lines to study glycyl tRNA synthetase (GARS) related neurodegeneration
J. Li1, C. Coxon1, S. Lee1, A. Goyenvalle1, G. Weir1, K. Talbot1, K. Davies1, Z. Cader1
1. DPAG, University of Oxford, Oxford, United Kingdom.
Background:GARS gene encodes the critical protein, Glycyl-tRNA Synthetase, which is an enzyme linking tRNA to glycine. Mutation in GARS have been shown to cause distal spinal muscular atrophy (dSMA) and Charcot-Marie-Tooth disease type 2D (CMT2D). In both conditions, the prominent early feature is wasting and weakness of the muscle of the hands and feet caused by degeneration of peripheral nerves (1). Two start codons in the Gars gene generate a short and a long version of the protein, which localize mainly to mitochondria and cytoplasm respectively. However, the function and relative role of the two isoforms in triggering neurodegeneration is unknown. The aim of this study is to identify the correlated genes differently expressed between wild type and mutant of the short and long Gars isoforms using a stable neuronal cell line model. Material and method:Lenti-XTM Tet-On® advanced inducible expression system was used to generate 4 types of mouse stable cell lines, with mouse cDNA: wildtype Gars and mutant_P278KY (2) in short or long Gars isoforms. 4 types of cDNA were tagged with an OneStrep tag and were cloned into pTightPuro vector. These were packaged into lentivirual particles and cotransduced with the regulator TetOn virus into target NSC-34 cells. We thereby obtained a system with Doxycycloine regulated expression of the gene of interest. Cells were tested with immunofluorescence and western blotting to confirm the expression of the transduced gene. RNAs were extracted from the expressed cell lines and were then carried out Affymetrix microarray study. Positive targets were validated using quantitative real-time PCR.Result: Immunofluorescence and western blot showed that NSC-34 cells were successfully transduced as indicated by the expression of the Strep tag. Using polyclonal cell lines, microarray data showed a few genes expressed differently between wildtype and mutant type with significant fold changes.
Where applicable, experiments conform with Society ethical requirements