A sequence-specific gene correction by an RNA-DNA oligonucleotide in mammalian cells characterized by transfection and nuclear extract using a lacZ shuttle system
O. Igoucheva et al., A sequence-specific gene correction by an RNA-DNA oligonucleotide in mammalian cells characterized by transfection and nuclear extract using a lacZ shuttle system, GENE THER, 6(12), 1999, pp. 1960-1971
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.