Abstract
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Pflügers Archiv: European Journal of Physiology |
| Vol/bind | 444 |
| Udgave nummer | 6 |
| Sider (fra-til) | 710-21 |
| Antal sider | 11 |
| ISSN | 0031-6768 |
| DOI | |
| Status | Udgivet - 2002 |
Bibliografisk note
Keywords: Animals; Biolistics; Glucose Transporter Type 4; Green Fluorescent Proteins; Indicators and Reagents; Luminescent Proteins; Male; Monosaccharide Transport Proteins; Muscle Contraction; Muscle Fibers; Muscle Proteins; Muscle, Skeletal; Plasmids; Protein Transport; Rats; Rats, Wistar; Recombinant Fusion Proteins; TransfectionAdgang til dokumentet
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Gene gun bombardment-mediated expression and translocation of EGFP-tagged GLUT4 in skeletal muscle fibres in vivo. / Lauritzen, Hans P M M; Reynet, Christine; Schjerling, Peter; Ralston, Evelyn; Thomas, Stephen; Galbo, Henrik; Ploug, Thorkil.
I: Pflügers Archiv: European Journal of Physiology, Bind 444, Nr. 6, 2002, s. 710-21.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review
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TY - JOUR
T1 - Gene gun bombardment-mediated expression and translocation of EGFP-tagged GLUT4 in skeletal muscle fibres in vivo.
AU - Lauritzen, Hans P M M
AU - Reynet, Christine
AU - Schjerling, Peter
AU - Ralston, Evelyn
AU - Thomas, Stephen
AU - Galbo, Henrik
AU - Ploug, Thorkil
N1 - Keywords: Animals; Biolistics; Glucose Transporter Type 4; Green Fluorescent Proteins; Indicators and Reagents; Luminescent Proteins; Male; Monosaccharide Transport Proteins; Muscle Contraction; Muscle Fibers; Muscle Proteins; Muscle, Skeletal; Plasmids; Protein Transport; Rats; Rats, Wistar; Recombinant Fusion Proteins; Transfection
PY - 2002
Y1 - 2002
N2 - Cellular protein trafficking has been studied to date only in vitro or with techniques that are invasive and have a low time resolution. To establish a gentle method for analysis of glucose transporter-4 (GLUT4) trafficking in vivo in fully differentiated rat skeletal muscle fibres we combined the enhanced green fluorescent protein (EGFP) labelling technique with physical transfection methods in vivo: intramuscular plasmid injection or gene gun bombardment. During optimisation experiments with plasmid coding for the EGFP reporter alone EGFP-positive muscle fibres were counted after collagenase treatment of in vivo transfected flexor digitorum brevis (FDB) muscles. In contrast to gene gun bombardment, intramuscular injection produced EGFP expression in only a few fibres. Regardless of the transfection technique, EGFP expression was higher in muscles from 2-week-old rats than in those from 6-week-old rats and peaked around 1 week after transfection. The gene gun was used subsequently with a plasmid coding for EGFP linked to the C-terminus of GLUT4 (GLUT4-EGFP). Rats were anaesthetised 5 days after transfection and insulin given i.v. with or without accompanying electrical hindleg muscle stimulation. After stimulation, the hindlegs were fixed by perfusion. GLUT4-EGFP-positive FDB fibres were isolated and analysed by confocal microscopy. The intracellular distribution of GLUT4-EGFP under basal conditions as well as after translocation to the plasma membrane in response to insulin, contractions, or both, was in accordance with previous studies of endogenous GLUT4. Finally, GLUT4-EGFP trafficking in quadriceps muscle in vivo was studied using time-lapse microscopy analysis in anaesthetised mice and the first detailed time-lapse recordings of GLUT4-EGFP translocation in fully differentiated skeletal muscle in vivo were obtained.
AB - Cellular protein trafficking has been studied to date only in vitro or with techniques that are invasive and have a low time resolution. To establish a gentle method for analysis of glucose transporter-4 (GLUT4) trafficking in vivo in fully differentiated rat skeletal muscle fibres we combined the enhanced green fluorescent protein (EGFP) labelling technique with physical transfection methods in vivo: intramuscular plasmid injection or gene gun bombardment. During optimisation experiments with plasmid coding for the EGFP reporter alone EGFP-positive muscle fibres were counted after collagenase treatment of in vivo transfected flexor digitorum brevis (FDB) muscles. In contrast to gene gun bombardment, intramuscular injection produced EGFP expression in only a few fibres. Regardless of the transfection technique, EGFP expression was higher in muscles from 2-week-old rats than in those from 6-week-old rats and peaked around 1 week after transfection. The gene gun was used subsequently with a plasmid coding for EGFP linked to the C-terminus of GLUT4 (GLUT4-EGFP). Rats were anaesthetised 5 days after transfection and insulin given i.v. with or without accompanying electrical hindleg muscle stimulation. After stimulation, the hindlegs were fixed by perfusion. GLUT4-EGFP-positive FDB fibres were isolated and analysed by confocal microscopy. The intracellular distribution of GLUT4-EGFP under basal conditions as well as after translocation to the plasma membrane in response to insulin, contractions, or both, was in accordance with previous studies of endogenous GLUT4. Finally, GLUT4-EGFP trafficking in quadriceps muscle in vivo was studied using time-lapse microscopy analysis in anaesthetised mice and the first detailed time-lapse recordings of GLUT4-EGFP translocation in fully differentiated skeletal muscle in vivo were obtained.
U2 - 10.1007/s00424-002-0862-5
DO - 10.1007/s00424-002-0862-5
M3 - Journal article
C2 - 12355170
VL - 444
SP - 710
EP - 721
JO - Pflügers Archiv - European Journal of Physiology
JF - Pflügers Archiv - European Journal of Physiology
SN - 0031-6768
IS - 6
ER -