Hasty P, Crist M, Grompe M, Bradley A
Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030.
Mol Cell Biol. 1994 Dec;14(12):8385-90. doi: 10.1128/mcb.14.12.8385-8390.1994.
We have analyzed the targeting frequencies and recombination products generated with isogenic vectors at the fah and fgr loci in embryonic stem cells. A single vector which could be linearized at different sites to generate either a replacement or an insertion vector was constructed for each locus. A replacement event predominated when the vectors were linearized at the edge of the homologous sequences, while an insertion event predominated when the vectors were linearized within the homologous sequences. However, the ratio of the targeting frequencies exhibited by the different vector configurations differed for the two loci. When the fgr vector was linearized as an insertion vector, the ratio of targeted to random integrations was four- to eightfold greater than when the vector was linearized as a replacement vector. By contrast, the ratio of targeted to random integrations at the fah locus did not vary with the linearization site of the vector. The different relationships between the targeting frequency and the vector configuration at the fgr and fah loci may indicate a DNA sequence or chromatin structure preference for different targeting pathways.
我们分析了在胚胎干细胞中，同基因载体在fah和fgr位点产生的靶向频率及重组产物。针对每个位点构建了一个可在不同位点线性化以产生置换载体或插入载体的单一载体。当载体在同源序列边缘线性化时，置换事件占主导；而当载体在同源序列内线性化时，插入事件占主导。然而，不同载体构型所表现出的靶向频率之比在两个位点有所不同。当fgr载体作为插入载体线性化时，靶向整合与随机整合的比例比载体作为置换载体线性化时大四至八倍。相比之下，fah位点靶向整合与随机整合的比例并不随载体的线性化位点而变化。fgr和fah位点靶向频率与载体构型之间的不同关系可能表明不同靶向途径对DNA序列或染色质结构存在偏好。
