title: 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 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.
authors.affiliation: Genetics Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, 104-0045, Tokyo, Japan.
authors.forename: Kazuteru
authors.initials: K
authors.lastname: Hatanaka
authors.forename: Shumpei
authors.initials: S
authors.lastname: Ohnami
authors.forename: Kimiko
authors.initials: K
authors.lastname: Yoshida
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Miura
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Aoyagi
authors.forename: Hiroki
authors.initials: H
authors.lastname: Sasaki
authors.forename: Masahiro
authors.initials: M
authors.lastname: Asaka
authors.forename: Masaaki
authors.initials: M
authors.lastname: Terada
authors.forename: Teruhiko
authors.initials: T
authors.lastname: Yoshida
authors.forename: Kazunori
authors.initials: K
authors.lastname: Aoki
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics* / Q000378:metabolism; D015723:Gene Library; D018014:Gene Transfer Techniques; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008957:Models, Genetic; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics; D013601:T-Lymphocytes / Q000378:metabolism; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins