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 conversion frequency rapidly and reproducibly. A shuttle vector was constructed to measure the frequency of targeted gene correction by RDO of the E. coli beta-galactosidase gene containing a single point mutation (G --> A), that resulted in inactivation of enzymatic activity. An RDO corrected the point mutation and restored the enzymatic activity, approximately 1%, determined by a histochemical staining in mammalian cells and by a color selection (blue or white) of bacteria transformed with Hirt DNA. In addition, we established an in vitro system capable of gene correction using nuclear extracts. CHO-K1 nuclear extracts corrected the point mutation approximately 0.1%, determined by bacterial transformation. Using the in vitro reaction, frequency of gene conversion in different cell types was measured. The embryonic fibroblasts from p53-/- mouse showed higher gene correction than that of the isogenic p53+/+ cells. Nuclear extracts from DT40 cells, which have a higher homologous recombination rate than any other mammalian cells exhibited 0.1-0.6% of gene correction. These results indicated that recombination may be rate-limiting in gene conversion by RDO in cells with competent mismatch repair activities. Utilizing transfection and in vitro reaction, we demonstrated that such a shuttle system might be useful in comparing the frequency of targeting among different cell types and to investigate the mechanism of gene conversion by RDO.
Igoucheva
O
O
Department of Dermatology and Cutaneous Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University and Jefferson Medical College, Philadelphia PA 19107, USA.
Peritz
A E
AE
Levy
D
D
Yoon
K
K
D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D015342:DNA Probes; D004789:Enzyme Activation; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D012150:Polymorphism, Restriction Fragment Length; D015347:RNA Probes; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics*
D015342:DNA Probes; D015347:RNA Probes; D001616:beta-Galactosidase