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PubMed · 12842439

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

Kazuteru Hatanaka·Shumpei Ohnami·Kimiko Yoshida·Yoshiaki Miura·Kazuhiko Aoyagi·Hiroki Sasaki·Masahiro Asaka·Masaaki Terada·Teruhiko Yoshida·Kazunori Aoki
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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 x 10(4) 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.

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AdenoviridaeBlotting, SouthernCell LineCloning, MolecularDNA, ComplementaryGene LibraryGene Transfer TechniquesGenetic TechniquesGreen Fluorescent ProteinsHumansLuminescent ProteinsModels, GeneticPhenotypePlasmidsPolymerase Chain ReactionRecombination, GeneticRetroviridaeT-LymphocytesTransfectionJournal ArticleResearch Support, Non-U.S. Gov't

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Kazuteru Hatanaka, Shumpei Ohnami, Kimiko Yoshida, Yoshiaki Miura, Kazuhiko Aoyagi, Hiroki Sasaki, Masahiro Asaka, Masaaki Terada, Teruhiko Yoshida, Kazunori Aoki. 2003. A simple and efficient method for constructing an adenoviral cDNA expression library.. https://doi.org/10.1016/s1525-0016(03)00138-2

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