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Published November 15, 2006 | public
Journal Article

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

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.

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.

Additional details

Created:
September 15, 2023
Modified:
October 23, 2023