A simple and efficient method for constructing an adenoviral cDNA expression library

Abstract

cDNA expression cloning is a powerful method for the identification of genes that are able to confer a selectable phenotype on specific cell types. An adenovirus vector is characterized by several advantages over plasmid DNA and retroviral vector-mediated gene transfer, such as broad host range and high infectivity. However, an expression cloning protocol using the adenovirus vector has not been reported. We describe here a simple and efficient method for constructing adenovirus cDNA expression libraries based on Cre-lox-mediated in vitro recombination between adenoviral shuttle plasmid cDNA libraries and adenoviral genomic DNA tagged with terminal protein. In a model experiment, EGFP clones present at the frequency of 0.003% in the shuttle plasmid library could be efficiently converted to adenoviral vector in a 6-cm dish under optimized conditions, indicating that high-complexity libraries harboring low-abundance cDNAs can be produced. The efficiency of this system was demonstrated by the isolation of cDNA for CD2 (frequency less than 1 in 0.3 × 104 transcripts in T cells) from the human T cells. This effective and versatile method can facilitate the functional identification of genes for a variety of purposes.