The X-gal caution in neural transplantation studies

Cell transplantation into host brain requires a reliable cell marker to tra ce lineage and location of grafted cells in host tissue. The lacZ gene enco des the bacterial (E. coli) enzyme beta -galactosidase (beta -gal) and is c ommonly visualized as a blue intracellular precipitate following its incuba tion with a substrate. "X-gal," in an oxidation reaction. LacZ is the "repo rter gene" most commonly employed to follow gene expression in neural tissu e or to track the fate of transplanted exogenous cells. If the reaction is not performed carefully-with adequate optimization and individualization of various parameters (e.g., pH, concentration of reagents, addition of chela tors, composition of fixatives) and the establishment of various controls-t hen misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from end ogenous nonbacterial beta -gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have emphasized how to recognize and to eliminat e these potential confounding artifacts in order to maximize the utility an d credibility of this histochemical technique as a cell marker. We briefly review the phenomenon in general, discuss a specific case that illustrates how an insufficiently scrutinized X-gal positivity can be a pitfall in cell transplantation studies, and then provide recommendations for optimizing t he specificity and reliability of this histochemical reaction for discernin g E. coli beta -gal activity.