Efficacy of Semliki Forest virus transduction of bovine adrenal chromaffin cells: an analysis of heterologous protein targeting and distribution.
In using chromaffin cells as a model for studying the mechanism of regulated exocytosis, there is a requirement for an efficient, safe, and robust system for the transduction and expression of heterologous cDNA in these cells. We have used Semliki Forest virus to transduce cDNAs encoding various proteins fused to enhanced green fluorescent protein (EGFP) into cultured bovine adrenal cells. Transduction is highly efficient but has no significant effect on the steady state levels of several endogenous proteins or of catecholamines in the transfected cells. Furthermore, the transfected cells show depolarization-induced calcium currents and nicotine-induced catecholamine release. We present data to show that virally transduced proteins are targeted to their intracellular locations correctly in chromaffin cells. The fusion protein pro-ANF-EGFP is specifically targeted to large dense-core vesicles as shown by its colocalization with acidophilic dyes and chromogranin A, making this a useful system for the study of secretory vesicle dynamics.
Duncan
Rory R
RR
University of Edinburgh, Division of Biomedical and Clinical Laboratory Sciences, Membrane Biology Group, Edinburgh EH8 9XD, Scotland. rd@srv4.med.ed.ac.uk
Greaves
Jennifer
J
Tapechum
Sompol
S
Apps
David K
DK
Shipston
Michael J
MJ
Chow
Robert H
RH
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D005089:Exocytosis; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012672:Semliki forest virus / Q000235:genetics*; D013997:Time Factors; D014161:Transduction, Genetic
D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins