title: Construction of a plasmid containing human SMN, the SMA determining gene, coupled to EGFP.
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.
authors: Laboratoire de Pathologie des Communications entre Cellules Nerveuses et Musculaires, EA 3429, Université Louis Pasteur, Illkirch, France.
authors: Anne-Sophie
authors: AS
authors: Arnold
authors: Mor
authors: M
authors: Gueye
authors: Philippe
authors: P
authors: Rondé
authors: Jean-Marie
authors: JM
authors: Warter
authors: Philippe
authors: P
authors: Poindron
authors: Jean-Pierre
authors: JP
authors: Gies
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein; D004352:Drug Resistance, Microbial / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009154:Mutation; D009355:Neomycin / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D055532:SMN Complex Proteins; D017422:Sequence Analysis, DNA; D014162:Transfection
chemical_list: D000900:Anti-Bacterial Agents; D017362:Cyclic AMP Response Element-Binding Protein; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D055532:SMN Complex Proteins; D049452:Green Fluorescent Proteins; D009355:Neomycin