Fig. 2.
Screening of a tagged full-length cDNA library. A tagged prostate cDNA library was created and cloned into the retrophage vector. The retrophage vector consists of retroviral as well as plasmid sequences within a λ phage backbone. Flanking LoxP sites allow for simple plasmid excision in Cre-expressing bacteria. Replication-incompetent virus was produced by transfecting retrophage DNA into AmphoPack-293 and entered into H1299. Total RNA was isolated 48 hr after infection and again after passaging for 7 days. Tags were rescued by RT-PCR, radiolabeled by 32P, and hybridized to replica nylon blots. Clones corresponding to those tags that disappeared on day 7 were isolated, recloned in a retroviral vector with no tags, and individually tested in colony assay. Inserts with confirmed biological activity were sequenced. One of them contained full-length p53 cDNA; this insert was radio-labeled and hybridized with a parallel nylon replica.