ADENOVIRUS TYPE 5 PARTICLES, CMV-Β-GALACTOSIDASE

This product is a concentrated source of highly-purified Adenovirus type 5 particles from a lysate of optimally-infected 293 cells, with an E1 and E3 deletion and insertion of CMV-driven nuclear-targeted β-galactosidase gene in E1 region. Double CsCl gradient purification with DNase treatment and dialysis, this Ad5 preparation is of very high quality and minimal lot-to-lot variation.

PRODUCT DETAILS – ADENOVIRUS TYPE 5 PARTICLES, CMV-Β-GALACTOSIDASE

• Adenovirus Type 5 Particles with CMV-induced β-Galactosidase.
• Double CsCl gradient purification with DNase treatment and dialysis.
• Produced in HEK293 cells and stored in pH 7.8 PBS buffer.

BACKGROUND

Adenoviruses are medium-sized (80–100 nm), non-enveloped viruses. They have an icosahedral nucleocapsid containing a linear, double-stranded DNA genome of approximately 36 kb (Nermut, 1984). The viral genome is grouped into different transcriptional units, designated early (E1, E2, E3, E4), intermediate, and late. The E1 gene is essential for activation of other viral genes and for viral replication. Deletion of the E1 gene results in viruses that are replication incompetent in normal cells. However, replication-competent viral particles can be produced from E1-deleted viral vectors by providing the E1 gene in trans.  The E3 gene is nonessential for either viral replication or infection (Flint, 1999).

Adenovirus type 5 is one of the most extensively studied and characterized adenoviruses and is the type used most frequently in generating recombinant adenoviruses for gene therapy. These vectors generally contain deletions of the E1 and E3 genes, which allows for insertion and packaging of up to 7.5 kb of foreign DNA, for gene delivery. This recombinant human adenovirus type 5 expresses β-galactosidase protein under the control of a CMV promoter. β-galactosidase is a glycoside hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides through the breaking of a glycosidic bond. An active enzyme may be detected using artificial chromogenic substrate 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside, X-gal. β-galactosidase will cleave the glycosidic bond in X-gal and form galactose and 5-bromo-4-chloro-3-hydroxyindole which dimerizes and oxidizes to 5,5′-dibromo-4,4′-dichloro-indigo, producing an intense blue precipitate. Therefore, this vector can be used to determine virus localisation, transduction efficiency and to optimize viral infection conditions in specific cell types.

REFERENCES

• Flint, J., 1999. Organization of the adenoviral genome. In: P. Seth, ed. Adenoviruses: Basic Biology to Gene Therapy. Austin, TX, USA: R.G. Landes Company, pp. 17-30.
• Nermut, M. V., 1984. The Architecture of Adenoviruses. In: . (eds) . In: H. S. Ginsberg, ed. The Adenoviruses. The Viruses. Boston, MA: Springer.