Amplification and expression of sequences cotransfected with a modular dihydrofolate reductase complementary DNA gene

Abstract

A modular dihydrofolate reductase (DHFR) gene has been constructed from a DHFR complementary DNA by the addition of proper transcription initiation and RNA splicing signals. After DNA transfection in the absence of carrier DNA this gene is capable of transforming Chinese hamster ovary cells deficient in DHFR (DHFR −) to the DHFR + phenotype. Initial transformants contain one to five copies of the modular gene integrated into the Chinese hamster ovary genome. After sequential increases in the concentration of methotrexate in the growth medium, cells are selected that contain up to 1000 copies of the transforming DNA. Since the entire early region of simian virus 40 (SV40) DNA was present intact in the original construct, it was possible to directly demonstrate that early proteins of SV40 are co-expressed after amplification. In one clone, a polypeptide related to SV40 small tumor antigen (t) represented over 10% of the total protein synthesis. The usefulness of this construct as a general amplification vector is discussed. Plasmid (pBR322) sequences, present intact in the amplified cellular DNA, have been rescued by bacterial transformation in order to demonstrate the integration of plasmid DNA into Chinese hamster ovary host DNA and also to demonstrate amplification of that Chinese hamster ovary host DNA after methotrexate selection. Characterization of an initial DHFR + transformant, its subclones, and first step methotrexate-resistant colonies from these subclones leads to the conclusion that (1) plasmid DNA integrated into Chinese hamster ovary DNA is extremely fluid, as demonstrated by its frequent deletion, and (2) transfected DNA sequences most frequently deleted are also those most frequently amplified on methotrexate selection.