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  • Title: Construction of a plasmid containing human SMN, the SMA determining gene, coupled to EGFP.
    Author: Arnold AS, Gueye M, Rondé P, Warter JM, Poindron P, Gies JP.
    Journal: Plasmid; 2002 Mar; 47(2):79-87. PubMed ID: 11982329.
    Abstract:
    We describe here the construction of plasmid pEGFP-C3/SMN, bearing the human SMN gene coupled to the green fluorescent protein (GFP) sequence. The mutation of the SMN gene is responsible for spinal muscular atrophy (SMA), a frequent human infantile genetic disease. We introduced the SMN cDNA into the multiple cloning site of pEGFP-C3. This plasmid bears the neomycin-resistance sequence and the enhanced green fluorescent protein (EGFP). It results in the expression of a fusion protein bearing SMN coupled to a carboxy-terminal GFP tag, used for fluorescence localization studies. Transfection of primary human myoblasts with pEGFP-C3 or pEGFP-C3/SMN revealed that EGFP is intracellularly localized within the cytosol as well as in the nucleus, while the fusion protein EGFP-SMN localized within the nucleus in prominent dot-like structures termed "gems." These data demonstrate that human primary muscle cells can be efficiently transfected and may have important implications for the development of therapeutic strategies in SMA.
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