X-gal

Vaccinia Virus as a Carrier of Vaccine Antigens

Published in F. Y. Liew, Vaccination Strategies of Tropical Diseases, 2017

Geoffrey L. Smith

The most widely used insertion site is the vaccinia TK gene since this provides a means of selection of recombinant (TK–) viruses. Selective methods are necessary since only 0.1% of the total virus from transfected cells is recombinant. The selection of TK– recombinants requires plaquing on TK– cells in the presence of 5-bromodeoxyuridine (BUdR). This restricts the cell lines that may be used and also causes generation of spontaneous TK– mutants which need to be distinguished from true TK– recombinants. Initially this was done by DNA hybridization using 32P-labeled foreign DNA as a probe,31 but subsequently new plasmid vectors have been designed which aid direct selection of recombinant viruses. These vectors contain two vaccinia promoters, one of which is used to drive expression of the desired foreign gene. The second promoter drives expression of a gene which aids selection of recombinants. For example, β-galactosidase expression can be used to enable visual selection of recombinants, since plaques formed by recombinants turn blue if incubated in the presence of X-gal.44 Expression of selectable genetic markers such as the gene conferring resistance to neomycin46 or the herpes simplex virus (HSV) TK gene in vaccinia TK– mutants also enable rapid selection of recombinant viruses.31,48,54

Long-Circulating Polymeric Nanoparticles for Drug and Gene Delivery to Tumors

Published in Mansoor M. Amiji, Nanotechnology for Cancer Therapy, 2006

Sushma Kommareddy, Dinesh B. Shenoy, Mansoor M. Amiji

The in vivo transfection efficiency of plasmid DNA-encapsulated (pCMV-β encoding β-galactosidase) PEGylated gelatin nanoparticles was evaluated by injecting these nanoparticles intravenously into LLC tumor-bearing female C57BL/6J mice. Following systemic administration, the animals were sacrificed, and the transgene expression in different organs was quantitatively determined by using o-nitrophenyl-β-d-galactopyranosidase (ONPG), a clear substrate of β-galactosidase that is converted to a yellow-colored product with an absorbance maximum at 420 nm, and qualitatively by X-gal® tissue staining. When compared to the gelatin nanoparticles, the PEGylated gelatin nanoparticles were found to efficiently transfect the tumor cells with the β-galactosidase expression increasing at time points up to 96 h post-transfection with the absorbance value at 420 nm increasing from 0.6 to 0.85 starting from 12 h post-transfection. The results of these studies clearly indicate that the long circulating, biocompatible, and biodegradable nanoparticles of PEGylated gelatin would be ideal for gene delivery applications to solid tumors.60

Published in Ronald M. Atlas, James W. Snyder, Handbook Of Media for Clinical Microbiology, 2006

Ronald M. Atlas, James W. Snyder

Use: For the identification of Salmonella species and differentiation of Salmonella spp. other organisms in the family Enterobacteriaceae. This medium combines two chromogens for the detection of Salmonella spp., 5-bromo-6-chloro-3-indolyl caprylate (Magenta-caprylate) and 5-bromo-4-chloro-3-In-dolyl β-D galactopyranoside (X-gal). X-gal is a substrate for the enzyme β-D-galactosidase. Hydrolysis of the chromogen, Mag-caprylate, by lactose negative Salmonella species results in magenta colonies. The medium contains bile salts to inhibit the growth of Gram-positive organisms and the addition of the selective supplement solution increases the selectivity of the medium. Novobiocin inhibits Proteus growth and cefsulodin inhibits growth of pseudo-monads.

Endothelial cell-derived extracellular vesicles alter vascular smooth muscle cell phenotype through high-mobility group box proteins

Published in Journal of Extracellular Vesicles, 2020

Michael J. Boyer, Yayoi Kimura, Tomoko Akiyama, Ariele Y. Baggett, Kyle J. Preston, Rosario Scalia, Satoru Eguchi, Victor Rizzo

Senescence-associated β galactosidase activity was measured using X-Gal staining system (Goldbio). In brief, VSMCs were serum-starved for 48 hours then incubated with EC EV or PBS for an additional 72 h. Cells were then rinsed twice with Hanks balanced salt solution containing calcium, fixed with a 4% paraformaldehyde solution in PBS for 10 min, washed again with the Hanks solution four times and incubated with 40 mmol/L phosphate buffer containing 1 mg/mL X-Gal in 150 mmol/L NaCl, 2 mmol/L MgCl2, 5 mmol/L K3Fe(CN)6 and 5 mmol/L K4Fe(CN)6 at 37°C for 12 h. Cells were washed twice with the Hanks solution with calcium and magnesium, and counter stained with Hoescht33342 for 5 min to stain nuclei. VSMC cultures were examined at 10x magnification to identify β galactosidase positive cells. Cells were counted in three to six individual wells per condition and values were expressed as a percentage of positive cells versus total cell number. Compiled data were expressed as fold-change between control and EV conditions.

Membrane biofouling behaviors at cold temperatures in pilot-scale hollow fiber membrane bioreactors with quorum quenching

Published in Biofouling, 2018

Kibaek Lee, Jun-Seong Park, Tahir Iqbal, Chang Hyun Nahm, Pyung-Kyu Park, Kwang-Ho Choo

The AHL QQ activity was evaluated using a bioassay based on the procedure described in a previous study (Yeon et al. 2008). Briefly, an indicating agar plate was prepared by mixing an overnight culture of A. tumefaciens A136 and LB agar at a volume ratio of 1:9. The indicating agar was supplemented with two antibiotics (tetracycline and spectinomycin) and X-gal (5-bromo-4-chloro-3-indolyl β-D-galactopyranoside). Then, each sample was placed on the indicating agar. The remaining AHLs were detected via the reporter strain (A136) with the production of β-galactosidase in response to AHLs, with β-galactosidase decomposing X-gal, resulting in a blue color. The AHL level was estimated quantitatively based on the size of blue color zones.

Effect of geraniol against arecoline induced toxicity in the third instar larvae of transgenic Drosophila melanogaster (hsp70-lacZ) Bg⁹

Published in Toxicology Mechanisms and Methods, 2019

Barkha Shakya, Sonam Shakya, Yasir Hasan Siddique

The results obtained for X-gal staining are also in concordance with the results obtained in ONPG assay. The larvae exposed to arecoline along with geraniol showed a dose dependent decrease in the X-gal reaction. The Escherichia coli Lac-Z gene encoding β-galactosidase is the classical histochemical reporter gene. The activity of β-galactosidase can be detected by using the substances which have galactose linked through a β-D-glycosidic linkage and one such substance is X-gal (Krasnow et al. 1991). The conserved nature of stress response across species allows the application of hsp based assays over a wide range of organisms and in this context the expression of hsp-70 has been suggested as a sensitive indicator of adverse biological effects (Varo et al. 2002; Bhargav et al. 2008). The results obtained for hsp-70 expression suggest that the geraniol is potent in reducing the expression of hsp-70. For measuring the extent of tissue damage in the third instar larvae induced by the exposure of arecoline along with geraniol the tissue explants were stained by trypan blue. It is a dye which is rapidly absorbed by dead or dying cells but is excluded from intact cell (Krebs and Feder 1997). A dose dependent decrease in the tissue damage was observed in the larvae exposed to arecoline along with geraniol. The induction of hsp-70 and the tissue damage has been correlated by exposing the third instar larvae at different temperature (Feder and Krebs 1998). In our present study the larvae exposed to 80 µM of arecoline along with 10, 20, 40, and 80 µM of geraniol showed a dose dependent decrease in the expression of hsp-70 as well as in the tissue damage.