Article Commentary: The X-gal Caution in Neural Transplantation Studies

Abstract

Cell transplantation into host brain requires a reliable cell marker to trace lineage and location of grafted cells in host tissue. The lacZ gene encodes the bacterial (E. coli) enzyme beta-galactosidase (beta-gal) and is commonly visualized as a blue intracellular precipitate following its incubation with a substrate, "X gal," in an oxidation reaction. LacZ is the "reporter gene" most commonly employed to follow gene expression in neural tissue 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 chelators, composition of fixatives) and the establishment of various controls--then misleading nonspecific background X-gal positivity can result, leading to the misidentification of cells. Some of this background results from endogenous nonbacterial beta-gal activity in discrete populations of neurons in the mammalian brain; some results from an excessive oxidation reaction. Surprisingly, few articles have empha sized how to recognize and to eliminate these potential confounding artifacts in order to maximize the utility and 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 the specificity and reliability of this histochemical reaction for discerning E. coli beta-gal activity.

Targeted Modification of Atrial Electrophysiology by Homogeneous Transmural Atrial Gene Transfer

“…Each sample was graded on a scale of 1 to 5 by blinded observers, as previously described. 12 There were no statistically significant differences in any histological parameters between treatment groups and control animals that underwent only open-chest manipulation of the heart. There was no association between fibrosis or mononuclear cell infiltration and the presence or type of virus, the presence of poloxamer, or the presence or concentration of trypsin, suggesting that the open-chest procedure rather than the gene transfer process caused the fibrosis.…”

“…4,11 X-gal staining was performed at pH 8 to minimize nonspecific staining. 12 The percentage of cells expressing ␤-galactosidase was determined by counting cells in 5 randomly selected microscopic sections for epicardial, midmyocardial, and endocardial layers (100 cells per field per layer; 500 cells per atrium for each layer). Histological analysis for inflammation, tissue architecture, and fibrosis was performed in a blinded manner, as previously described.…”

Optimizing Techniques for Tracking Transplanted Stem Cells In Vivo

“…invitrogen.com). It has been known for years that a wide variety of mammalian cells possess significant B-gal activity, which is expressed at higher levels in the liver, kidney, pancreas, spleen, uterus, thyroid, and intestines than in other tissues [30][31][32]. Thus, the appearance of a chromogenic or fluorescent product of B-gal cleavage simply indicates that cleavage occurred.…”

“…1E). Some groups were misled by these normal variations in endogenous B-gal activity and first reported and then subsequently retracted their conclusions that BMDCs had replaced large groups of CNS neurons [32]. Thus, a discussion of its optimal use is in order.…”

“…1D, 1F). Techniques to optimize B-gal-based tracking systems in the CNS have been reviewed recently [32].…”