Adeno-cosmid cloning vectors for regulated gene expression.
BACKGROUND
Adenovectors are widely used for efficient delivery of genes into a variety of cell types and organisms. However, the construction of the desired vector/genes combination, especially if it involves the cloning of several gene cassettes, can be laborious due to the large size of these vectors. New methods are needed to simplify the construction of complex combinations of gene cassettes into adenovectors.


METHODS
Using simple cloning techniques and exploiting the lambda-phage packaging system, we devised efficient methods for the 'selection' of the desired vector constructs. Thus we generated a series of cosmids containing the adeno helper dependent (HD) backbone in which we inserted cis- and trans-acting tetracycline (tet) elements for the regulation of any gene of interest. One of these cosmids has been used to produce an HD adenovirus carrying a tetracycline-regulated gene expressing beta-galactosidase.


RESULTS
We have demonstrated that the adeno-cosmid system allows rapid and efficient cloning of genes of interest in helper dependent vectors, and described a prototype 'ready-to-use' vector in which any gene of interest can be easily expressed under the control of the tet system. The HD viruses produced with this novel methodology can be grown at high titers, can be easily separated from the helper adenovirus, and allow delivery and regulated gene expression in a variety of tissues.


CONCLUSIONS
Exploiting the lambda-packaging system, complex adeno constructs can be generated with a simple and reproducible protocol, which allows selection of the desired size construct, counterselecting for the frequently observed intramolecular recombinations and deletions.
Giampaoli
Saverio
S
IRBM P. Angeletti, via Pontina km 30.600, 00040 Pomezia (Roma), Italy.
Nicolaus
Giancarlo
G
Delmastro
Paola
P
Cortese
Riccardo
R
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D003360:Cosmids / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics*; D006378:Helper Viruses / Q000235:genetics; D006624:Hippocampus / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D019076:Transgenes; D001616:beta-Galactosidase / Q000235:genetics
D001616:beta-Galactosidase