GENE TARGETING IN RAT EMBRYO FIBROBLASTS PROMOTED BY THE POLYOMAVIRUSLARGE T-ANTIGEN

We used the recombination-promoting activity of the polyomavirus large T antigen (T-ag) to increase the frequency of gene targeting in rat f ibroblasts. We constructed a cell line carrying a functional polyomavi rus replication origin and a transformation-defective middle T-ag onco gene. The structure of the locus was such that homologous recombinatio n with the targeting DNA reconstituted a functional transforming gene and converted the cells from the normal to the transformed state. Intr oduction of the large T-ag with the targeting DNA promoted recombinati onal events that corrected the mutation in either the target locus or the targeting DNA. The frequency of recombination was not substantiall y influenced by the extent of homology between the recombining sequenc es. However, it was reduced when the replication origin was inactivate d in the targeting DNA, and was reduced further when the origin was in activated in the target locus.