End extension repair of introduced targeting vectors mediated by homologous recombination in mammalian cells.
We have studied the mechanism of targeted recombination in mammalian cells using a hemizygous adenine phosphoribosyltransferase-deficient (APRT-) Chinese hamster ovary (CHO) cell mutant as a recipient. Three structurally different targeting vectors with a 5' or a 3', or both, end-deleted aprt sequence, in either a closed-circular or linear form, were transfected to the cells with a mutated aprt gene by electroporation. APRT-positive (APRT+) recombinant clones were selected and analyzed to study the gene correction events of the deletion mutation. Some half of 58 recombinant clones obtained resulted from corrections of the deleted chromosomal aprt gene by either gene replacement or gene insertion, a mechanism which is currently accepted for homologous recombination in mammalian cells. However, the chromosomal sequence in the remaining half of the recombinants remained uncorrected but their truncated end of the aprt gene in the incoming vectors was corrected by extending the end beyond the region of homology to the target locus; the corrected vector was then randomly integrated into the genome. This extension, termed end extension repair, was observed with all three vectors used and was as far as 4.6-kilobase (kb) or more long. It is evident that the novel repair reaction mediated by homologous recombination, in addition to gene replacement and gene insertion, is also involved in gene correction events in mammalian cells. We discuss the model which may account for this phenomenon.
Aratani
Y
Y
Kihara Institute for Biological Research, Yokohama City University, Japan.
Okazaki
R
R
Koyama
H
H
D000228:Adenine Phosphoribosyltransferase / Q000172:deficiency / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004260:DNA Repair; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic; D017384:Sequence Deletion; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D004247:DNA; D000228:Adenine Phosphoribosyltransferase