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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.

journal_name

Dev Biol

journal_title

Developmental biology

authors

Damle S,Hanser B,Davidson EH,Fraser SE

doi

10.1016/j.ydbio.2006.06.016

subject

Has Abstract

pub_date

2006-11-15 00:00:00

pages

543-50

issue

2

eissn

0012-1606

issn

1095-564X

pii

S0012-1606(06)00918-3

journal_volume

299

pub_type

杂志文章

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