Secretion from bovine chromaffin cells acutely expressing exogenous proteins using a recombinant Semliki Forest virus containing an EGFP reporter.
Acute expression of recombinant proteins throughout a population of postmitotic bovine chromaffin cells was achieved using the Semliki Forest virus expression system (P. Liljestrom and H. Garoff (1991) Biotechnology 9:1356-1361). The virus was modified to express a green fluorescent protein, which faithfully reported the expression of the recombinant proteins. Two types of reporting virus were constructed: the first included a second subgenomic element, and the second an internal ribosome entry site. Both were used to express the recombinant proteins beta-galactosidase, 5HT3 receptor, or tetanus toxin light chain. Beta-galactosidase was used to quantify the rate of expression of recombinant protein in chromaffin cells, the 5HT3 receptor to trigger secretion, and the toxin to block secretion. The experiments clearly show that infection and expression of recombinant proteins throughout a population of chromaffin cells do not, per se, affect the rate and extent of triggered exocytosis, endocytosis, or membrane recycling pathways. The catecholamine content of the cell is unaltered, and the secretory mechanism can be accessed within a few hours after infection. This noncytopathic method of acutely expressing specific proteins at physiological levels in chromaffin cells offers a powerful new tool for dissecting the roles of many proteins implicated in exo- and endocytosis.
Knight
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DE
Medical Research Council Laboratory for Molecular Cell Biology, University College London, United Kingdom.
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D019439:Chromaffin Cells / Q000166:cytology* / Q000502:physiology*; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007668:Kidney; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D011985:Receptors, Serotonin / Q000096:biosynthesis / Q000235:genetics; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins / Q000096:biosynthesis; D012672:Semliki forest virus; D013744:Tetanus Toxin / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D002395:Catecholamines; D008164:Luminescent Proteins; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D011994:Recombinant Proteins; D013744:Tetanus Toxin; D049452:Green Fluorescent Proteins; D007770:L-Lactate Dehydrogenase; D001616:beta-Galactosidase; D002118:Calcium