A simplified system for generating recombinant adenoviruses

A simplified system for generating recombinant adenoviruses

^*The Howard Hughes Medical Institute, ^†The Program in Human Genetics and Molecular Biology, ^‡The Johns Hopkins Oncology Center, 424 North Bond Street, Baltimore, MD 21231

Contributed by Bert Vogelstein

Abstract

Recombinant adenoviruses provide a versatile system for gene expression studies and therapeutic applications. We report herein a strategy that simplifies the generation and production of such viruses. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria rather than in eukaryotic cells. After transfections of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Homogeneous viruses can be obtained from this procedure without plaque purification. This system should expedite the process of generating and testing recombinant adenoviruses for a variety of purposes.

Footnotes

↵ § To whom reprint requests should be addressed. e-mail: tche{at}welchlink.welch.jhu.edu.
ABBREVIATIONS:

β-gal,

β-galactosidase;

CMV,

cytomegalovirus;

efu,

expression-forming units;

GFP,

green fluorescent protein;

ITR,

inverted terminal repeat