Isolation of Temperature-Sensitive Mutants of L-Cells

Archived with permission from the National Academy of Sciences. This article was originally published in Proceedings of the National Academy of Sciences (PNAS) Volume 66 Issue 2. Please refer to www.pnas.org. The author holds copyright to this article.Procedures are described for the isolation of conditional lethal mutants of mouse L-60T cells. The mutant lines were temperature sensitive by the following criteria: (a) colony-forming ability, (b) growth in suspension culture, and (c) rate of uptake of tritiated-thymidine. The development and use of conditional lethal mutations has considerably facilitated the analysis of function in viruses and bacteria; as a result, the main features of regulation in these materials have been outlined. However, it is not yet clear how applicable these models of regulation are to eucaryotic cells and, in particular, whether or not they provide a basis for understanding the processes involved in the differentiation of mammalian cells. Although techniques have been developed for genetic studies with somatic cell hybrids, progress in this area has been severely inhibited by the limited numbers and kinds of mutants available. Drug resistant lines and a few auxotrophs have been reported, and the only other major source of mutants is through the establishment of cell lines derived from the somatic tissues of patients with known genetically determined defects. If techniques were developed for the isolation of conditional lethal mutants of somatic mammalian cells, it should be possible to obtain a much broader spectrum of mutations. In this paper we describe the isolation of temperature- sensitive mutants of mouse L-cells.Supported by the Medical Research Council of Canada and the National Cancer Institute of Canad