Transformation of chicken embryo fibroblasts by direct DNA transfection of single oncogenes: comparative analyses of src, erbB, myc, and ras.
Chicken embryo fibroblasts (CEF) have been used extensively to study the transformation parameters of a number of avian sarcoma-leukemia viruses. Previously, oncogene transformation of CEF has been conducted almost exclusively with replicating viruses, because of perceived difficulties with direct DNA transfection. Here, we show that CEF can be efficiently and stably transfected by selection for the neomycin resistance gene (neo). Cotransfection of neo with various oncogenes resulted in CEF transformation in vitro and, in several instances, sarcoma formation in vivo. Transfection of src, myc, erbB, and ras, either singly or in combination, resulted in soft-agar colonies with unique morphologies. Transfection of a family of v-src, c-src, and v/c-src chimeric constructs demonstrated the ability of the assay to discriminate between transforming and nontransforming genes. Transfection of a number of erbB variants showed that internal mutations, primarily in the kinase domain, contribute significantly to the ability to transform fibroblasts. The tumorigenic potential detected by transfection of oncogenes faithfully reproduced those previously reported by using viral infections. Our studies establish the utility of CEF transformation by direct DNA transfection. This method should prove useful in analyzing oncogenes, (e.g., myc) that do not readily transform rodent cell lines and in studying host-range mutants of oncogenes, such as those recently identified for src and erbB.
Antczak
M
M
Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
Kung
H J
HJ
D000818:Animals; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D002678:Chimera; D005347:Fibroblasts / Q000378:metabolism / Q000382:microbiology; D011905:Genes, ras; D009368:Neoplasm Transplantation; D009857:Oncogenes; D010957:Plasmids; D012190:Retroviridae / Q000235:genetics; D013045:Species Specificity; D014162:Transfection