Novel use of a selectable fusion gene as an “In‐Out” marker for studying genetic loss in mammalian cells

Abstract

Recent demonstrations of loss of heterozygosity in a wide variety of human cancers suggest that large multilocus genetic deletions (presumably including tumor suppressor genes) constitute a major class of genetic alteration in human carcinogenesis. Here we show that a bifunctional fusion gene (Hytk), suitable for both positive and negative selection, is an effective marker for studying genetic loss in mammalian cells with minimal interference from point-mutational changes. Studies with a transgenic V79 cell line in which a single functional copy of Hytk was stably inserted into the genome in a retroviral vector showed that loss of the marker (and presumably flanking cellular genetic material) could be induced efficiently by ionizing radiation (gamma-rays and fast neutrons) but only weakly by the powerful point-mutagen benzo[a]pyrene diol epoxide. In a first application of the system, we provide evidence that radiation-induced loss can occur through an indirect mechanism after a high-frequency event. Collectively, our results suggest that the Hytk marker should be a valuable tool for studying genome position effects on the tolerance of genetic loss in cultured human cells that represent different stages in clonal evolution and tumor progression.

X-ray-induced mutations in mouse embryonic stem cells

“…Parallel spontaneous mutations were compared with the treated population and any mutation that was present in the exposed and spontaneous mutants was assumed to be a pre-existing mutation and only counted as one independent spontaneous mutation. (27)(28)(29)(30)(31). Of these loci, the Hprt locus has been most commonly used as a target because both forward and reverse selection are possible.…”

Functional evidence from microcell‐mediated chromosome transfer of myeloid leukemia suppressor genes on human chromosomes 7 and 11

“…Spleens were removed from euthanized mice and DNA was extracted from leukemic cells for further analysis (Clark et al, 1996). DNA from hybrid clones and leukemic cells from mice were screened by PCR for the presence of the hygromycin phosphotransferase gene (Hpt) using the procedure of Trott et al (1995).…”

Selection for retroviral insertions into regulated genes

“…This report introduces two technical modifications that extend the feasibility of this strategy for the in vitro identification of regulated genes. By combining gene trapping with efficient selection schemes based on fluorescence-activated cell sorting (FACS) (25) or dominant positive and negative drug selection (20,34), a strategy that facilitates the identification and cloning of genes with regulated expression and that could be useful in the study of a variety of cell lines was developed. As an example, we applied this approach to the identification of integrations into genes regulated by a transcription factor.…”

“…However, in the NIH 3T3 cells (a tetraploid cell line) used in the experiments presented here, only 1% of the G418-resistant cells stained positive with X-Gal. In pROSAHyTK the reporter gene encodes a fusion protein with both hygromycin phosphotransferase activity, which confers resistance to hygromycin, and thymidine kinase activity, which confers sensitivity to ganciclovir (20,34). Selection for proviral integration is provided by the neo gene under the control of the PGK gene promoter.…”