A highly efficient transfection protocol and a plasmid vector for the construction and delivery into mammalian cells of cDNA expression libraries have been developed. Many commonly used fibroblastic cell lines are stably transfected at frequencies of greater than 10% with the cDNA cloning vector that incorporates a neo selectable marker. The system permits cloning of cDNAs on the basis of their function expressed in mammalian cells. The majority of human cancers seem to be induced by a recessive mechanism (recessive oncogenes). To clone such a novel type oncogene, nitroso methylurea-transformed BHK cells were transfected with a cDNA expression library constructed with the new vector and mRNA prepared from primary human fibroblast cells. After neo selection and morphological screening, two flat revertants that were unable to grow in soft agar were isolated. In secondary transfection with the genomic DNA prepared from one of the two revertants, the flat phenotype co-transmitted with neo resistance. The cDNA that induces flat reversion is being recovered in E. coli for molecular cloning and characterization.
