When goldfish erythrophores isolated from the skin by tissue digestion and centrifugation in a Percoll density gradient were transfected in a monolayer-culture with v-Ha-ras or v-src oncogene either singly or in combination with v-myc by means of calcium phosphate-DNA co-precipitation, there appeared a certain number of transformants manifesting a chromatoblast-like profile and tumorous phenotypes as seen in the capability for unlimited growth, and piling-up in a monolayer-culture or colony formation in semi-solid soft agar. After successive growth in vitro for longer than one month which was scarcely observed with the erythrophores, the vast majority of such transformants began to differentiate into erythrophores and ceased proliferation spontaneously. The onset of their differentiation was ascertained by the deposition of marker pteridine pigments. None of the transformants differentiated into melanophores or iridophores or other neural crest derivatives as seen in goldfish erythrophoroma cells. Little difference was observed in their transforming efficiency (0.2-0.3 transformants/μg DNA) between the combinations of oncogenes applied but a tendency was noted that cells transfected with ras or src in combination with myc developed the capacity to grow for a longer period and differentiated at a later stage than those transfected solely with ras or src. One cell line (ESM-1) derived from the erythrophores transfected with src/myc grew successively over nine months, indicating its acquisition of immortality. The expression of the transfected oncogenes in this cell line was examined in comparison with the erythrophoroma cells by Western and Northern blot analyses.
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