Note to users. If you're seeing this message, it means that your browser cannot find this page's style/presentation instructions -- or possibly that you are using a browser that does not support current Web standards. Find out more about why this message is appearing, and what you can do to make your experience of our site the best it can be.

Subscribe

Sci. STKE, 14 October 2003
Vol. 2003, Issue 204, p. pl12
[DOI: 10.1126/stke.2003.204.pl12]

PROTOCOLS

Coupling the Iron-Responsive Element to GFP--An Inducible System to Study Translation in a Single Living Cell

Bernhard Goetze*, Barbara Grunewald, Michael A. Kiebler, and Paolo Macchi*

Max-Planck-Institute for Developmental Biology, Spemannstrasse 35, D-72076 Tübingen, Germany.

Abstract: Local protein synthesis in a cell represents an elegant mechanism to achieve important biological phenomena such as cell migration, body axis formation during embryonic development and establishment of cell polarity. A prerequisite to studying translation in a restricted cellular compartment is the ability to unambiguously discriminate between proteins that arise through local protein synthesis and those that reach the site of interest by diffusion or transport. To tackle this problem, we set up a green fluorescent protein (GFP)-based reporter system that allows one to uncouple the translation of reporter gene mRNA from its subcellular localization. The system is based on the iron-responsive element, which regulates the translation of both endogenous ferritin and transferrin transcripts in response to changes in iron concentration. Translation of the reporter messenger RNA (mRNA) is thus dependent on iron in the medium; both its transcription and localization, however, are unaffected. Known targeting sequences can be used to direct the mRNA transcript to a subcellular compartment of interest. For instance, the full-length 3'-untranslated region of calcium/calmodulin-dependent protein kinase IIα mRNA can be added to the construct, after the stop codon of the GFP sequence, to selectively target the transcript into the dendrites of transiently transfected hippocampal neurons. This novel fluorescent assay will allow us to address a number of important biological questions in living mammalian cells.

*Corresponding authors. Telephone, 0049-7071-601-329; fax, 0049-7071-601-305; e-mail,bernhard.goetze{at}tuebingen.mpg.de; paolo.macchi{at}tuebingen.mpg.de

Citation: B. Goetze, B. Grunewald, M. A. Kiebler, P. Macchi, Coupling the Iron-Responsive Element to GFP--An Inducible System to Study Translation in a Single Living Cell. Sci. STKE 2003, pl12 (2003).

Read the Full Text



To Advertise     Find Products


Science Signaling. ISSN 1937-9145 (online), 1945-0877 (print). Pre-2008: Science's STKE. ISSN 1525-8882