Development of a highly efficient expression cDNA cloning system: application to oncogene isolation.

TLDR

The authors developed a high‑complexity, unidirectionally inserted cDNA cloning system that enables efficient plasmid rescue. They applied this system to construct a cDNA library from an NIH 3T3 transformant induced by mouse hepatocellular carcinoma DNA and used it to transfect NIH 3T3 cells. Transfection yielded multiple transformed foci from which identical plasmids with transforming activity were rescued; sequence analysis revealed the oncogene arose from recombination between an unknown gene and the mouse B‑raf homolog, and its recurrence in independent transformants indicates tumor‑derived origin, underscoring the system’s high efficiency and broad applicability for isolating biologically relevant cDNAs.

Abstract

We developed an expression cDNA cloning system capable of generating high-complexity libraries with unidirectionally inserted cDNA fragments and allowing efficient plasmid rescue. As an application of this system, a cDNA library was constructed from an NIH 3T3 transformant induced by mouse hepatocellular carcinoma DNA. Transfection of NIH 3T3 cells by the library DNA led to the detection of several transformed foci from which identical plasmids with transforming ability could be rescued. Structure and sequence analysis of the cDNA clones revealed that the oncogene was created by recombinational events involving an unknown gene and the mouse homologue of the B-raf protooncogene. Detection of the same genetic rearrangement in independent primary transformants implied that generation of the oncogene occurred within the tumor rather than during DNA transfection or cDNA library construction. The high frequency at which clones were identified and the large sizes of some of the transforming cDNA inserts isolated suggest wide applicability of this mammalian expression cloning system for isolating cDNAs of biologic interest.