Francès V, Bastin M
Department of Biochemistry, Université de Sherbrooke, Sherbrooke, Quebec, Canada.
Nucleic Acids Res. 1996 Jun 1;24(11):1999-2004. doi: 10.1093/nar/24.11.1999.
We used the recombination-promoting activity of the polyomavirus large T antigen (T-ag) to increase the frequency of gene targeting in rat fibroblasts. We constructed a cell line carrying a functional polyomavirus replication origin and a transformation-defective middle T-ag oncogene. The structure of the locus was such that homologous recombination with the targeting DNA reconstituted a functional transforming gene and converted the cells from the normal to the transformed state. Introduction of the large T-ag with the targeting DNA promoted recombinational events that corrected the mutation in either the target locus or the targeting DNA. The frequency of recombination was not substantially influenced by the extent of homology between the recombining sequences. However, it was reduced when the replication origin was inactivated in the targeting DNA, and was reduced further when the origin was inactivated in the target locus.
我们利用多瘤病毒大T抗原（T-ag）的重组促进活性来提高大鼠成纤维细胞中基因靶向的频率。我们构建了一个携带功能性多瘤病毒复制起点和转化缺陷型中T-ag癌基因的细胞系。该基因座的结构使得与靶向DNA的同源重组能够重构一个功能性的转化基因，并将细胞从正常状态转变为转化状态。将大T-ag与靶向DNA一起导入可促进重组事件，从而纠正靶基因座或靶向DNA中的突变。重组频率并未受到重组序列之间同源性程度的实质性影响。然而，当靶向DNA中的复制起点失活时，重组频率会降低，而当靶基因座中的复制起点失活时，重组频率会进一步降低。
