Construction of a plasmid containing human SMN, the SMA determining gene, coupled to EGFP.
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.
Arnold
Anne-Sophie
AS
Laboratoire de Pathologie des Communications entre Cellules Nerveuses et Musculaires, EA 3429, Université Louis Pasteur, Illkirch, France.
Gueye
Mor
M
Rondé
Philippe
P
Warter
Jean-Marie
JM
Poindron
Philippe
P
Gies
Jean-Pierre
JP
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
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