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Quantitative imaging of cis-regulatory reporters in living embryos

Dmochowski, Ivan J. and Dmochowski, Jane E. and Oliveri, Paola and Davidson, Eric H. and Fraser, Scott E. (2002) Quantitative imaging of cis-regulatory reporters in living embryos. Proceedings of the National Academy of Sciences of the United States of America, 99 (20). pp. 12895-12900. ISSN 0027-8424. PMCID PMC130556. https://resolver.caltech.edu/CaltechAUTHORS:20111019-092516176

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Abstract

A confocal laser scanning microscopy method has been developed for the quantitation of green fluorescent protein (GFP) as a reporter of gene activity in living three-dimensional structures such as sea urchin and starfish embryos. This method is between 2 and 50 times more accurate than conventional confocal microscopy procedures depending on the localization of GFP within an embryo. By using coinjected Texas red dextran as an internal fluorescent standard, the observed GFP intensity is corrected for variations in laser excitation and fluorescence collection efficiency. To relate the recorded image intensity to the number of GFP molecules, the embryos were lysed gently, and a fluorometric analysis of their contents was performed. Confocal laser scanning microscopy data collection from a single sea urchin blastula required less than 2 min, thereby allowing gene expression in dozens of embryos to be monitored in parallel with high spatial and temporal resolution.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1073/pnas.202483199 DOIArticle
http://www.pnas.org/content/99/20/12895PublisherArticle
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC130556/PubMed CentralArticle
ORCID:
AuthorORCID
Fraser, Scott E.0000-0002-5377-0223
Additional Information:© 2002 by the National Academy of Sciences. Contributed by Eric H. Davidson, August 9, 2002. Published online before print September 17, 2002. We thank Drs. Andrew Cameron and Chiou-Hwa Yuh for many helpful discussions; Carolina B. Livi for providing the Krox-GFP construct; Dr. Veronica Hinman for providing starfish embryos; and the California Institute of Technology Seismological Laboratory for computational facilities. This work was supported by the Helen Hay Whitney Foundation (to I.J.D.), National Aeronautics and Space Administration Earth System Science Fellowship Grant NGT5-30393 (to J.E.D.), and National Institutes of Health Grant HD37105 (to E.H.D. and S.E.F.).
Funders:
Funding AgencyGrant Number
Helen Hay Whitney FoundationUNSPECIFIED
NASA Earth System Science FellowshipNGT5-30393
NIHHD37105
Issue or Number:20
PubMed Central ID:PMC130556
Record Number:CaltechAUTHORS:20111019-092516176
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20111019-092516176
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:27299
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:24 Oct 2011 22:41
Last Modified:03 Oct 2019 03:22

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