A simple and efficient method for constructing an adenoviral cDNA expression library.
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.
Hatanaka
Kazuteru
K
Genetics Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, 104-0045, Tokyo, Japan.
Ohnami
Shumpei
S
Yoshida
Kimiko
K
Miura
Yoshiaki
Y
Aoyagi
Kazuhiko
K
Sasaki
Hiroki
H
Asaka
Masahiro
M
Terada
Masaaki
M
Yoshida
Teruhiko
T
Aoki
Kazunori
K
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
D018076:DNA, Complementary; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins