Viral infection protocols.
This chapter describes the protocol for preparation of recombinant adenoviruses and infection of target cells to express transiently G-protein-coupled receptors or other proteins of interest. Adenoviruses are nonenveloped viruses containing a linear double-stranded DNA genome. Their life cycle does not normally involve integration into the host genome, rather they replicate as episomal elements in the nucleus of the host cell and consequently there is no risk of insertional mutagenesis. The wild-type adenovirus genome is approx 35 kb, of which up to 30 kb can be replaced by foreign DNA. Adenoviral vectors are very efficient at transducing the gene of interest in target cells in vitro and in vivo and can be produced at high titers (>10(11)/mL). The viral infection has a number of useful features: (1) the efficiency of gene transduction is very high (up to 100% in sensitive cells). (2) The infection is easy and does not alter physically the cell membrane for gene transduction. (3) It is possible to infect cells that are resistant to transfection with plasmids (including nondividing cells).
Porcellini
Antonio
A
Department of Molecular Pathology, INM Neuromed, IRCCS, Pozzilli, Italy.
De Blasi
Antonio
A
D000256:Adenoviridae; D000818:Animals; D005822:Genetic Vectors; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic / Q000379:methods*
D043562:Receptors, G-Protein-Coupled