Abstract
Photoconvertible fluorescent proteins, such as Kaede, can be switched irreversibly from their native color to a new one. This property can be exploited to visualize de novo mRNA translation, because newly synthesized proteins can be distinguished from preexisting ones by their color. In this protocol, Kaede cDNA linked to the 3′ untranslated region (UTR) of β-actin is delivered into cells fated to become the retina by injection into Xenopus blastomeres. Brief exposure (6–10 s) to UV light (350–410 nm) of Kaede-positive retinal axons/growth cones efficiently converts Kaede from its native green fluorescence to red. The reappearance of the green signal reports the synthesis of new Kaede protein. This approach can be used to investigate the spatiotemporal control of translation of specific mRNAs in response to external stimuli and to test the efficiency of full-length versus mutant UTRs. The 3-d protocol can be adapted for broad use with other photoactivatable fluorescent proteins.
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Acknowledgements
We thank M. Spira for suggesting the use of Kaede. We also thank M. Agathocleous, J. Falk, L. Leung, A. Lin and F. van Horck for critical reading of the manuscript. This work was supported by a Croucher Scholarship (K.-M.L.) and a Wellcome Trust Programme Grant (C.E.H.).
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Leung, KM., Holt, C. Live visualization of protein synthesis in axonal growth cones by microinjection of photoconvertible Kaede into Xenopus embryos. Nat Protoc 3, 1318–1327 (2008). https://doi.org/10.1038/nprot.2008.113
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DOI: https://doi.org/10.1038/nprot.2008.113
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