The admid system : Generation of recombinant adenoviruses by Tn7-mediated transposition in E. coli

• Published in: BioTechniques Vol. 29; no. 1; pp. 146 - 154
• Main Authors: RICHARDS, C. A, BROWN, C. E, COGSWELL, J. P, WEINER, M. P
• Format: Journal Article
• Language: English
• Published: Natick, MA Eaton 01.07.2000; Taylor & Francis Group
• Subjects: Adenoviridae - genetics; Adenoviridae - isolation & purification; Adenovirus; beta-Galactosidase - genetics; Biological and medical sciences; Biotechnology; Cell Line; DNA Transposable Elements; DNA, Recombinant; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene therapy; Genetic Vectors; Health. Pharmaceutical industry; Humans; Industrial applications and implications. Economical aspects; Plasmids - genetics; Polymerase Chain Reaction; Transfection; transposon Tn7; Virus; Plasmid; Adenoviridae; Recombinant virus; Escherichia coli; Transposon; Bacteria; Method; Gene therapy; Vector; Enterobacteriaceae
• ISSN: 0736-6205; 1940-9818
• DOI: 10.2144/00291rr01

A new system has been developed for generating recombinant adenoviruses by Tn7-mediated transposition in E. coli. Low copy number E. coli plasmids containing a full-length adenoviral genome with lacZattTn7 replacing E1 have been constructed. The adenovirus plasmid or admid, as well as high copy number progenitors, were stably maintained in E. coli strain DH10B. Several transfer vectors containing a mammalian expression cassette flanked by Tn7R and Tn7L were used as donors to transpose the mini-Tn7 into the E1 region of the adenoviral genome. Transposed recombinant admids are readily identified by their beta-galactosidase phenotype. Transfection of admid DNA into producer cells resulted in the efficient production of infectious adenovirus. This easy-to-use, efficient system generates pure, clonal stocks of recombinant adenovirus without successive rounds of plaque purification.