Histochemical techniques for locating Escherichia coli beta-galactosidase activity in transgenic organisms.
Escherichia coli beta-galactosidase is a commonly used reporter molecule for analyzing gene expression. Recently, beta-galactosidase fusions have been applied to a variety of eukaryotic systems. The techniques for constructing and introducing beta-galactosidase fusion constructs as well as soluble assays for total enzyme function have been described in detail elsewhere. This article describes histochemical techniques for analyzing organisms that contain a functional beta-galactosidase fusion construct. The object is to determine semiquantitatively which cells are expressing the beta-galactosidase fusion protein, as well as the subcellular localization of the protein. Due to its prevalence in the author's laboratory, Caenorhabditis elegans is used as a canonical organism for the detailed methods described.
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Department of Embryology, Carnegie Institution of Washington, Baltimore, MD 21210.
D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology*; D002863:Chromogenic Compounds; D004926:Escherichia coli / Q000201:enzymology*; D005697:Galactosides; D016591:Histocytological Preparation Techniques; D007211:Indoles; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000302:isolation & purification* / Q000378:metabolism
D002863:Chromogenic Compounds; D005697:Galactosides; D007211:Indoles; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside