The X-gal caution in neural transplantation studies.
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.
Sanchez-Ramos
J
J
Department of Neurology, University of South Florida, Tampa 33612, USA. jsramos@com1.med.usf.edu
Song
S
S
Dailey
M
M
Cardozo-Pelaez
F
F
Hazzi
C
C
Stedeford
T
T
Willing
A
A
Freeman
T B
TB
Saporta
S
S
Zigova
T
T
Sanberg
P R
PR
Snyder
E Y
EY
D000818:Animals; D016477:Artifacts; D001921:Brain / Q000378:metabolism; D002863:Chromogenic Compounds / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005404:Fixatives; D005697:Galactosides / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D006651:Histocytochemistry / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000378:metabolism / Q000637:transplantation*; D051381:Rats; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
D002863:Chromogenic Compounds; D005404:Fixatives; D005697:Galactosides; D007211:Indoles; D001616:beta-Galactosidase; C044888:5-bromo-4-chloro-3-indolyl beta-galactoside