Histochemical staining of clonal mammalian cell lines expressingE. coli ? galactosidase indicates heterogeneous expression of the bacterial gene

Abstract

An evaluation has been made of the E. coli beta-galactosidase (beta-gal) gene for use as a reporter gene in mammalian cells in culture. We have adopted a histochemical procedure which enables identification of those cells within a population that express the introduced bacterial gene. Data is presented concerning the sensitivity of the histochemical method relative to an immunological method of detection. It has been found that several clonal cell lines generated after transfection of human 293 cells with a Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter-beta-gal construction are mosaic for expression of the introduced mini-gene. Furthermore, after treatment of these clonal cell lines with the nucleoside analog 5-aza-cytidine (5-aza-C), an increase in production of beta-gal under control of this promoter element was observed.

β-Galactosidase marker genes to tag and track human hematopoietic cells

“…Treatment with a demethylating agent such as 5Ј-azacytidine increases the percentage of X-Gal ϩ cells. This observation and others 28 suggest that DNA methylation may partly explain the decrease in ␤-gal expression in transduced cells. No morphological (Wright-Giemsa staining) or proliferative (growth curve kinetics, tritiated thymidine analysis, and the percentage of cells in different phases of the cell cycle) differences were observed between nls-LacZ-expressing and nonexpressing cells.…”

“…Staining with 5-bromo-4-chloro-3-indolyl-␤-D-galactoside (X-Gal), which involves the degradation of X-Gal into galactose and a blue-indigo chromogene product, is the most widely used assay for the detection of transduced cells. 28 It is worth noting that cells have to be fixed before addition of the substrate. X-Gal degradation can be inhibited by 4-␤-D-galacto-D-glucose.…”

TnIfast IRE enhancer: Multistep developmental regulation during skeletal muscle fiber type differentiation

“…TnILacZ1 contains 530 bp of 5Ј-flanking DNA, exon 1, intron 1, and the first (untranslated) part of exon 2 of the quail TnIfast gene (Baldwin et al, 1985), linked to a 5.2-kb SmaI/ EcoRI fragment of pRSVZ (MacGregor et al, 1987) containing promoterless E. coli LacZ sequences and SV40 splicing and polyadenylation sequences (see construct maps in Fig. 1).…”

Intercellular trafficking of VP22-GFP fusion proteins is not observed in cultured mammalian cells

“…Bacterial ␤-galactosidase was detected by histochemical staining with X-gal as described previously. 20 Automatic DNA sequencing was performed at the DNA Core Sequencing Facility at the University of Texas MD Anderson Cancer Center.…”