A sequence-specific gene correction by an RNA-DNA oligonucleotide in mammalian cells characterized by transfection and nuclear extract using a lacZ shuttle system

The variability in gene conversion frequency by an RNA-DNA oligonucleotide (RDO) prompted us to develop a system as a means of measuring the conversio n frequency rapidly and reproducibly. A shuffle vector was constructed to m easure the frequency of targeted gene correction by RDO of the E. coil beta -galactosidase gene containing a single point mutation (G --> A), that resu lted in inactivation of enzymatic activity. An RDO corrected the point muta tion and restored the enzymatic activity, approximately 1%, determined by a histochemical staining in mammalian cells and by a color selection (blue o r white) of bacteria transformed with Hirt DNA. In addition, we established an in vitro system capable of gene correction using nuclear extracts. CHO- KI nuclear extracts corrected the point mutation approximately 0.1%, determ ined by bacterial transformation. Using the in vitro reaction, frequency of gene conversion in different cell types was measured. The embryonic fibrob lasts from p53-/- mouse showed higher gene correction than that of the isog enic p53+/+ cells. Nuclear extracts from DT40 cells, which have a higher ho mologous recombination rate than any other mammalian cells exhibited 0.1-0. 6% of gene correction. These results indicated that recombination may be ra te-limiting in gene conversion by RDO in cells with competent mismatch repa ir activities. Utilizing transfection and in vitro reaction, we demonstrate d that such a shuttle system might be useful in comparing the frequency of targeting among different cell types and to investigate the mechanism of ge ne conversion by RDO.