Using reporter genes to study cis-regulatory elements.
This chapter summarizes four powerful assays for analyzing gene expression in cis-regulatory studies. The enzymatic assays (CAT, luciferase, lacZ) are currently limited by their application to embryo homogenates or fixed samples, but offer more robust analysis of gene activity than GFP. Assays based on CAT enzymatic activity or on CAT mRNA detection by WMISH are laborious but are well established for accurately quantifying gene expression and to determine spatial patterns at defined timepoints during development. LacZ assays are the current standard for spatially visualizing gene products in whole-mount fixed embryos. They are very sensitive but they provide limited temporal or quantitative information due to the perdurance of beta-galactosidase and the subtleties of the staining technique. Recently developed luciferase assays promise to be even more sensitive and accurate than the CAT and lacZ assays, and applicable to living cells and embryos. But, they have not yet been well established in invertebrate deuterostome research. GFP allows visualization of gene expression within living embryos. But because this is not an enzymatic assay, sensitivity can be a problem, particularly for weak promoters. Furthermore, imaging live embryos and quantifying gene expression in space and time (due to scattering of light by tissue, the perdurance of GFP, and other experimental details) is currently fraught with challenges. Ongoing improvements in imaging technology and the advent of multiple fluorescent proteins, as well as fluorescent and luminescent assays for vital imaging, will dramatically facilitate studies of gene expression in the coming decade.
Arnone
Maria I
MI
Stazione Zoologica Anton Dohrn, Villa Cornunale, 80121 Napoli, Italy.
Dmochowski
Ivan J
IJ
Gache
Christian
C
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D005809:Genes, Regulator / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D012617:Sea Urchins / Q000235:genetics* / Q000378:metabolism
D049452:Green Fluorescent Proteins; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase