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In vitro gene manipulation of spinal muscular atrophy fibroblast cell line using gene-targeting fragment for restoration of SMN protein expression

Gene Therapy volume 23, pages 10–17 (2016)Cite this article

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Abstract

The reduced level of survival motor neuron (SMN) protein, caused by homozygous deletions in the SMN gene, led to a common neurodegenerative disorder known as spinal muscular atrophy (SMA). In spite of extensive efforts to find a cure for SMA, there is currently no effective treatment available for this devastating disease. In this study, restoration of SMN expression through ‘gene-targeting’ method in SMA fibroblast cells was attempted. We designed a 2697-bp gene-targeting cassette; it consisted of an SMN1 open reading frame expressing 38 kD SMN protein and the upstream and downstream regions of exon 1 of SMN1 gene at the ends as the homology arms. SMA fibroblast cells were transfected by gene-targeting cassette using Lipofectamine LTX-PLUS reagent. Occurrence of homologous recombination in selected cells was investigated by PCR analysis. Increased expression of SMN protein was shown by real-time PCR and western blotting analysis. The immunofluorescence analysis results demonstrated that the number of SMN nuclear structures, Gems, was the same as or greater than the number of Gems found in normal fibroblasts. The results of this study indicate that gene-targeting methods do, in fact, present as an alternative for restoration of SMN expression in SMA patients-derived cells in vitro.

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Acknowledgements

We thank the Ege University Scientific Research Projects (APAK) for financial support (Grant Number 2012/TIP/033). We also express our sincere appreciation to Professor Dr Bahram Kazemi (Director of Medical Biotechnology Department, School of Medicine, Shahid Beheshti University of Medical Sciences) for assistance in this study.

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Authors and Affiliations

  1. Department of Biotechnology, Graduate School of Natural and Applied Sciences, Ege University, Izmir, Turkey

    A Rashnonejad

  2. Young Researchers and Elites Club, North Tehran Branch, Islamic Azad University, Tehran, Iran

    A Rashnonejad

  3. Department of Medical Biology, Ege University Faculty of Medicine, Izmir, Turkey

    C Gündüz & S Y Süslüer

  4. Department of Medical Genetics, Ege University Faculty of Medicine, Izmir, Turkey

    H Onay, B Durmaz & F Özkınay

  5. Department of Biotechnology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    M Bandehpour

  6. Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    M Bandehpour

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  1. A Rashnonejad
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Correspondence to A Rashnonejad.

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Rashnonejad, A., Gündüz, C., Süslüer, S. et al. In vitro gene manipulation of spinal muscular atrophy fibroblast cell line using gene-targeting fragment for restoration of SMN protein expression. Gene Ther 23, 10–17 (2016). https://doi.org/10.1038/gt.2015.92

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