Differential spontaneous transformation in vitro of newly established mouse fibroblast lines carrying or lacking the viable yellow mutation (Avy) of the mouse agouti locus
✍ Scribed by Wen-luang Wendy Hsiao; George L. Wolff; Beverly M. North; Michael M. Ollmann; Gregory S. Barsh; Hung Fan
- Book ID
- 102656954
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 979 KB
- Volume
- 15
- Category
- Article
- ISSN
- 0899-1987
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✦ Synopsis
The pleiotropic effects of the viable yellow mutation (A""), an allele of the mouse agouti coat-color locus, include increased susceptibility t o spontaneous and chemically induced tumors that affect a wide variety of tissues. As a first step toward understanding the molecular basis of this phenomenon, we established permanent fibroblast-like cell lines from newborn Aw/a and control congenic a h mice and compared their growth characteristics in vitro. From the VYMfffC3HflNctr and YS/WffCH3flNctr-Aw inbred strains, each of which carries theAwallele on a congenic background, 38 clonalAw/a and 16 clonalala lines were established. Regardless of inbred strain, all Av/a cell lines exhibited a significant degree of spontaneous transformation, as assessed by focus formation in monolayer culture, whereas none of the a h cell lines formed foci in prolonged cultures. To test whether changes in dosage of the A"-or a-bearing chromosomes were related t o these events, we analyzed each cell line with a closely linked molecular probe from the Emv-75 locus, which in the VY strain detects a restriction fragment length variant (RFLV) informative for the A"-and a-bearing chromosomes. Most of the transformed foci maintained heterozygosity for RFLVs detected by the probe, but two of the transformants lost the a-associated RFLV, and at least one of the transformants exhibited amplification of the Aw-associated RFLV. When the transformants were analyzed with 5' sequences derived from the recently cloned agouti gene, three of eight transformants lost the a-associated RFLV, and two of the transformants showed amplification of the A"-associated RFLV. Reverse transcriptase-polymerase chain reaction assays indicated that agouti RNA was detected in Awh, not a h cell lines. Surprisingly, some of the Av/a transformants lacked agouti RNA. These results suggest that deregulated expression of the A" allele is required for the initiation but not for the maintenance of transformation of the Aw/a cell cultures. These cell lines may provide an in vitro culture system for studying the effect of the agouti gene on tumorigenicity as well as t o potentially study other pleiotropic phenotypes.