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Confocal quantification of cis-regulatory reporter gene expression in living sea urchin

Damle, Sagar and Hanser, Bridget and Davidson, Eric H. and Fraser, Scott E. (2006) Confocal quantification of cis-regulatory reporter gene expression in living sea urchin. Developmental Biology, 299 (2). pp. 543-550. ISSN 0012-1606. https://resolver.caltech.edu/CaltechAUTHORS:20160121-135401587

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Abstract

Quantification of GFP reporter gene expression at single cell level in living sea urchin embryos can now be accomplished by a new method of confocal laser scanning microscopy (CLSM). Eggs injected with a tissue-specific GFP reporter DNA construct were grown to gastrula stage and their fluorescence recorded as a series of contiguous Z-section slices that spanned the entire embryo. To measure the depth-dependent signal decay seen in the successive slices of an image stack, the eggs were coinjected with a freely diffusible internal fluorescent standard, rhodamine dextran. The measured rhodamine fluorescence was used to generate a computational correction for the depth-dependent loss of GFP fluorescence per slice. The intensity of GFP fluorescence was converted to the number of GFP molecules using a conversion constant derived from CLSM imaging of eggs injected with a measured quantity of GFP protein. The outcome is a validated method for accurately counting GFP molecules in given cells in reporter gene transfer experiments, as we demonstrate by use of an expression construct expressed exclusively in skeletogenic cells.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/j.ydbio.2006.06.016DOIArticle
http://www.sciencedirect.com/science/article/pii/S0012160606009183PublisherArticle
ORCID:
AuthorORCID
Fraser, Scott E.0000-0002-5377-0223
Additional Information:© 2006 Elsevier Inc. Received for publication 10 February 2006; revised 8 June 2006; accepted 9 June 2006. Available online 14 June 2006. This work was supported by NIH grant HD-37105 and Department of Energy grant DE-FG02-03ER63584. Confocal images were taken at the Biological Imaging Center in the Beckman Institute at the California Institute of Technology.
Funders:
Funding AgencyGrant Number
NIHHD-37105
Department of Energy (DOE)DE-FG02-03ER63584
Subject Keywords:Confocal laser scanning microscopy; GFP; Sea urchin cis-regulation
Issue or Number:2
Record Number:CaltechAUTHORS:20160121-135401587
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160121-135401587
Official Citation:Sagar Damle, Bridget Hanser, Eric H. Davidson, Scott E. Fraser, Confocal quantification of cis-regulatory reporter gene expression in living sea urchin, Developmental Biology, Volume 299, Issue 2, 15 November 2006, Pages 543-550, ISSN 0012-1606, http://dx.doi.org/10.1016/j.ydbio.2006.06.016. (http://www.sciencedirect.com/science/article/pii/S0012160606009183)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:63850
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:22 Jan 2016 22:29
Last Modified:03 Oct 2019 09:32

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