Confocal quantification of cis-regulatory reporter gene expression in living sea urchin.
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.
Damle
Sagar
S
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Hanser
Bridget
B
Davidson
Eric H
EH
Fraser
Scott E
SE
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*
D049452:Green Fluorescent Proteins