title: 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.
authors.affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Sagar
authors.initials: S
authors.lastname: Damle
authors.forename: Bridget
authors.initials: B
authors.lastname: Hanser
authors.forename: Eric H
authors.initials: EH
authors.lastname: Davidson
authors.forename: Scott E
authors.initials: SE
authors.lastname: Fraser
mesh_terms: D000818:Animals; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism; D005775:Gastrula / Q000166:cytology / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D018613:Microscopy, Confocal; D012617:Sea Urchins / Q000166:cytology / Q000196:embryology / Q000378:metabolism*
chemical_list: D049452:Green Fluorescent Proteins