Intracellular delivery of a Tat-eGFP fusion protein into muscle cells.
The Tat protein from HIV-1, when fused with heterologous proteins or peptides, can traverse biological membranes in a process called "protein transduction," delivering its cargo into cells. A Tat-eGFP fusion protein was purified from bacteria to study the transduction kinetics of Tat fusion proteins into cultured myoblasts and in the muscle tissue. Correctly folded Tat-eGFP reaches a maximum intracellular level in nearly 30 min, while its endogenous fluorescence is first detected only after 14 h. The nuclear localization signal from the basic domain of Tat was not sufficient to confer nuclear localization to Tat-eGFP, suggesting that the nuclear import pathway used by the exogenously added Tat-eGFP might be sensitive to the folding state of eGFP. In mice, the direct delivery to the muscle tissue using subcutaneous injections or the intra-arterial pathway led to few positive fibers in the muscle periphery or surrounding the blood vessels. Muscles injected with Tat-eGFP showed intense labeling of the extracellular matrix (ECM), suggesting that, although Tat fusion proteins can transduce muscle fibers, their binding by components of the ECM surrounding myofibers could interfere with the intracellular transduction process.
Unité de Recherche en Génétique Humaine, Centre de Recherche du Centre Hospitalier de l'Université laval, Ste-Foy, Quebec, Canada G1V 4G2.
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Caron
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Torrente
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Camirand
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Bujold
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Chapdelaine
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Leriche
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Bresolin
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Tremblay
D000818:Animals; D023181:Antimicrobial Cationic Peptides; D001692:Biological Transport; D002478:Cells, Cultured; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D015497:HIV-1; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D018482:Muscle, Skeletal / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage; D015533:Transcriptional Activation; D014161:Transduction, Genetic; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D023181:Antimicrobial Cationic Peptides; D015696:Gene Products, tat; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins