Afrooz Rshnonejad, Cumhur Gündüz, Sunde Süslüer, Hüseyin Onay, Burak Durmaz, Mojgan Bandehpour and Ferda Özkinay
Department of Biotechnology, Ege University, Izmir, Turkey
The reduced level of Survival motor neuron (SMN) protein causes a neurodegenerative disorder named as Spinal muscular atrophy (SMA) that characterized by progressive paralysis and symmetrical muscle weakness. Majority of SMA patients have homozygous deletions in SMN gene. Despite extensive efforts to find a cure for SMA, there is still no effective treatment for this devastating disease. In the present study, 'gene targeting' method based on homologous recombination was used to restoration of SMN protein expression in SMA type I fibroblasts. Gene targeting fragment including SMN1 cDNA and homolog upstream and downstream sequences was designed and subcloned into a pGH vector. SMA type I fibroblast cell line (Coriel institue, GM03813) was transfected with DNA fragments using Lipofectamine LTX Plus Reagent (invitrogen, 15338). Single cell colony was achieved by serial dilution and PCR analysis was performed to confirm the occurrence of homologous recombination in transfected cells. Restoration of SMN protein expression was confirmed by RT-PCR and Western Blot analysis. The immunofluorescent results demonstrated localization of SMN protein in the nucleus of transfected cells. Our result show that homologous recombination could present as an alternative method for in vitro restoration of SMN protein. In vitro correction of patients stem cell by homologous recombination and transplantation of these cells to patients could be investigated to find alternative treatment for SMA disease.
Keywords: Survival motor neuron protein, spinal muscular atrophy, Homologous recombination, Gene targeting.