Berinstein N, Pennell N, Ottaway C A, Shulman M J
Department of Medicine, University of Toronto, Ontario, Canada.
Mol Cell Biol. 1992 Jan;12(1):360-7. doi: 10.1128/mcb.12.1.360-367.1992.
Homologous recombination is now routinely used in mammalian cells to replace endogenous chromosomal sequences with transferred DNA. Vectors for this purpose are traditionally constructed so that the replacement segment is flanked on both sides by DNA sequences which are identical to sequences in the chromosomal target gene. To test the importance of bilateral regions of homology, we measured recombination between transferred and chromosomal immunoglobulin genes when the transferred segment was homologous to the chromosomal gene only on the 3' side. In each of the four recombinants analyzed, the 5' junction was unique, suggesting that it was formed by nonhomologous, i.e., random or illegitimate, recombination. In two of the recombinants, the 3' junction was apparently formed by homologous recombination, while in the other two recombinants, the 3' junction as well as the 5' junction might have involved a nonhomologous crossover. As reported previously, we found that the frequency of gene targeting increases monotonically with the length of the region of homology. Our results also indicate that targeting with fragments bearing one-sided homology can be as efficient as with fragments with bilateral homology, provided that the overall length of homology is comparable. The frequency of these events suggests that the immunoglobulin locus is particularly susceptible to nonhomologous recombination. Vectors designed for one-sided homologous recombination might be advantageous for some applications in genetic engineering.
同源重组如今在哺乳动物细胞中被常规用于用转移的DNA替换内源性染色体序列。传统上构建用于此目的的载体，使得替换片段两侧都有与染色体靶基因中的序列相同的DNA序列。为了测试同源性双侧区域的重要性，我们测定了转移的和染色体免疫球蛋白基因之间的重组情况，此时转移片段仅在3'端与染色体基因同源。在分析的四个重组体中的每一个中，5'连接处都是独特的，这表明它是由非同源重组形成的，即随机或非法重组。在两个重组体中，3'连接处显然是由同源重组形成的，而在另外两个重组体中，3'连接处以及5'连接处可能都涉及非同源交叉。如先前报道的那样，我们发现基因靶向频率随同源区域长度单调增加。我们的结果还表明，只要同源性的总长度相当，用具有单侧同源性的片段进行靶向与用具有双侧同源性的片段一样有效。这些事件的频率表明免疫球蛋白基因座特别容易发生非同源重组。设计用于单侧同源重组的载体可能在基因工程的某些应用中具有优势。
