An efficient method for infection of adrenal chromaffin cells using the Semliki Forest virus gene expression system.
We have expanded the use of the Semliki Forest virus (SFV) by infecting chromaffin cells with synaptic proteins at high efficiency. Using the SFV gene expression system, up to 40% of cultured bovine chromaffin cells express the protein of interest within 12-48 h after infection. In order to learn about the basic physiological properties of infected cells, we performed membrane capacitance measurements using the whole-cell patch-clamp technique and monitored catecholamine release with amperometry. We found that chromaffin cells infected with green fluorescent protein (GFP) were comparable to control cells in intracellular calcium concentrations ([Ca2+]i), leak currents and cell sizes. In response to depolarization, calcium currents were elicited and the cells secreted catecholamine. Comparison of the calcium current amplitude and the size of the readily releasable pool of vesicles revealed a small decrease in these parameters compared to control cells. The refilling kinetics after pool depletion, however, were not altered. Overexpressed munc13-1 translocates to the plasma membrane in response to phorbol esters, an effect that is also observed in fibroblasts transfected with conventional methods. Thus, the use of the SFV gene expression system to infect chromaffin cells represents a major improvement in infection efficiency compared to other methods. It opens up new opportunities to introduce synaptic proteins into chromaffin cells and study their role in secretion.
Ashery
U
U
Max-Planck-Institute for Biophysical Chemistry, Department of Membrane Biophysics, Göttingen, Germany.
Betz
A
A
Xu
T
T
Brose
N
N
Rettig
J
J
D000311:Adrenal Glands / Q000821:virology*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D019439:Chromaffin Cells / Q000166:cytology / Q000378:metabolism* / Q000821:virology*; D004594:Electrophysiology; D015870:Gene Expression; D005821:Genetic Techniques; D007239:Infections; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D012672:Semliki forest virus / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D002118:Calcium