Cytochemical detection of gene expression: Light and electron microscope evaluation of bacterial β-galactosidase reaction product

Abstract

Recombinant DNA technology allows the introduction of genes into the genome of cells in vitro and in vivo. “Reporter” genes cotransfected with other genes of interest indicate successful transfection because cellular expression can be detected by histochemistry, autoradiography, or in situ hybridization histochemistry. The Escherichia coli gene, lacZ, is a frequently used reporter gene because it does not affect the usual functions or activities of the cell; the gene is present and expressed in successive generations; and bacterial β-galactosidase (β-gal), the enzyme produced as a result of expression of the gene, can be detected cytochemically. Studies that evaluate transfected cells among mixed cell populations in vivo, e.g. gene therapy and cell lineage studies, can benefit from the detectability of the cytochemical reaction product at the light and electron microscope levels. After incubation at pH 7.4 in X-gal (5-bromo-4-chloro-3 indolyl-β-D-galactoside), a commercially available indigogenic substrate, a blue-green reaction product results in cells that have synthesized functional bacterial β-galactosidase enzyme. This reaction product is visible in intact cells, and is retained during processing and embedment for thin section analysis in the transmission electron microscope. This report demonstrates the value of lacZ as a reporter gene using a clone of transfected HTD114 cells of human fibrosarcoma origin, selected for their high lacZ expression. Figure 1 is a dark field image of reaction product in 1 μm thick epon sections of cells that were incubated in X-gal, fixed, pelleted, osmicated, dehydrated, and embedded by routine methods. Figure 2 shows the appearance of the reaction product at the ultrastructural level. At both LM and EM levels, the reaction product varies in quantity among cells; appears focally concentrated in some cells, diffuse in others; and is confined to the cytosome with a noticeable ring-like distribution around the nucleus in yet other cells. Dark field analysis of sections is especially helpful for detecting relatively low quantities of reaction product. The distinctive ultrastructural appearance of the reaction product allows the unequivocal identification of cells expressing lacZ as well as the ultrastructural features of the cells. Ultrastructural characterization of the cell is often important for precise identification of cell type, especially in tissues such as lung in which there are many different cell types.