DNA fragments from F9 PyEC mutants increase expression of heterologous genes in transfected F9 cells

Abstract

Restriction fragments encompassing the DNA sequence changes, which allow polyoma mutants to productively infect embryonal carcinoma (EC) cells, have been coupled to the Herpes simplex virus thymidine kinase (HSV TK) gene and to the bacterial chloramphenicol acetyl transferase (CAT) gene. F9 TK(−) EC cells have been transfected with the TK constructions and transformation to TK(+) colonies has been determined. F9 EC cells, retinoic-acid-induced differentiating F9 cultures, mouse fibroblasts, and mouse myoblasts have been transfected with the CAT constructions and CAT enzyme activity has been measured from the transfected cells. The results suggest that the mutant changes function at the level of gene-expression enhancement, the mutant constructions increasing the TK transformation frequency and also yielding a higher level of CAT enzyme activity when compared with constructions having no polyoma fragment or the analogous wildtype polyoma restriction fragment. The sequence specificity for enhancement changes upon differentiation of F9 EC cells, with the CAT enzyme levels from the wild-type construction approaching the values obtained from a PyEC mutant construction having a single base-pair difference.