Elsevier

Developmental Biology

Volume 299, Issue 2, 15 November 2006, Pages 543-550
Developmental Biology

Genomes & Developmental Control
Confocal quantification of cis-regulatory reporter gene expression in living sea urchin

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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.

Abbreviations

CLSM
confocal laser scanning microscopy
GFP
green fluorescent protein
QPCR
quantitative real-time PCR
GRN
gene regulatory network
WMISH
whole mount in situ hybridization
hpf
hours postfertilization

Keywords

Confocal laser scanning microscopy
GFP
Sea urchin cis-regulation

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