Isolation of a line of immortal chicken embryo fibroblasts after transfection with the nuclei of Rous sarcoma virus-transformed Chinese hamster cells

Abstract

Secondary cultures of chicken embryo fibroblasts were transfected with purified nuclei from lysed cells of a clonal line of temperature-sensitive Rous sarcoma virus ( tsRSV)-transformed Chinese hamster fibroblasts. After propagation for 3 months an established cell line designated ChR32 was obtained in one chicken cell culture. The cells of this line have been propagated so far for 18 months, whereas normal chicken embryo fibroblasts died after 2 months. The established cells were heteroploid with a diploid modal number of macrochromosomes and two Z chromosomes. No Chinese hamster chromosomes could be identified. Southern blot analysis of DNA from the uncloned ChR32 cells and the clones provided evidence that these established cells were, in fact, clonal in origin and contained full-length RSV proviruses and no defective proviruses. Furthermore, they contained, at the 3′ end proviral-cellular junction, BglII, HpaI, KpnI, SacI, and XbaI fragments of the same size as the Chinese hamster donor cells, suggesting that the cellular sequence adjacent to the provirus is of Chinese hamster origin. The cells after establishment were able to grow continuously at 37 ° or 41 °C and produce a large amount of ts sarcoma virus particles. A corollary finding was that these virus particles were non-leaky for the transforming function at the non-permissive temperature.