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

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.06.016 Byline: Sagar Damle (a), Bridget Hanser (a)(b), Eric H. Davidson (a), Scott E. Fraser (a)(b)(c) Keywords: Confocal laser scanning microscopy; GFP; Sea urchin cis-regulation 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 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. Author Affiliation: (a) Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA (b) Beckman Institute, California Institute of Technology, Pasadena, CA 91125, USA (c) Division of Engineering and Applied Science, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 10 February 2006; Revised 8 June 2006; Accepted 9 June 2006