END EXTENSION REPAIR OF INTRODUCED TARGETING VECTORS MEDIATED BY HOMOLOGOUS RECOMBINATION IN MAMMALIAN-CELLS
END EXTENSION REPAIR OF INTRODUCED TARGETING VECTORS MEDIATED BY HOMOLOGOUS RECOMBINATION IN MAMMALIAN-CELLS
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DOI：
10.1093/nar/20.18.4795
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发表时间：
1992-09-25
影响因子：
14.9
通讯作者：
KOYAMA, H
中科院分区：
文献类型：
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作者：
ARATANI, Y;OKAZAKI, R;KOYAMA, H
我们使用半合子腺嘌呤磷酸核糖转移酶缺陷（APRT-）中国仓鼠卵巢（CHO）细胞突变体作为受体，研究了哺乳动物细胞中的靶向重组机制。通过电穿孔将具有5'或3'或两者末端删除的aprt序列（呈闭环或线性形式）的三种结构不同的靶向载体转染至具有突变的aprt基因的细胞中。选择并分析APRT阳性（APRT+）重组克隆，以研究缺失突变的基因校正事件。获得的 58 个重组克隆中约有一半是通过基因替换或基因插入对删除的染色体 aprt 基因进行修正而产生的，这是目前公认的哺乳动物细胞同源重组机制。然而，剩余一半重组体中的染色体序列仍未校正，但通过将末端延伸到与目标基因座同源的区域之外来校正传入载体中aprt基因的截短末端；然后将校正后的载体随机整合到基因组中。这种延伸被称为末端延伸修复，在使用的所有三个载体中均观察到，并且长度长达 4.6 千碱基 (kb) 或更长。显然，除了基因替换和基因插入之外，同源重组介导的新型修复反应也参与哺乳动物细胞中的基因校正事件。我们讨论可以解释这种现象的模型。
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 structually 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.