Thyagarajan B, Johnson B L, Campbell C
Department of Pharmacology, University of Minnesota Medical School, Minneapolis 55455, USA
Nucleic Acids Res. 1995 Jul 25;23(14):2784-90. doi: 10.1093/nar/23.14.2784.
We evaluate the effect of target site transcription on gene targeting in cultured human fibrosarcoma cells. A number of cell lines that harbored a plasmid recombination substrate within their chromosomal DNA were created. Gene targeting frequency was then measured at these different loci in the presence and absence of an agent that stimulated target site transcription. We observed that gene targeting was significantly enhanced by RNA transcription. The magnitude of transcription-stimulated gene targeting varied from 3-fold to > 20-fold. No increase in gene targeting was observed, however, when transcription proceeded away from, rather than through, the recombination site. Transcription-stimulated gene targeting was also observed when single-stranded plasmid vectors complementary to either the coding or template strand were used as recombination substrates. Our results indicate that gene targeting, like other forms of DNA recombination, can be stimulated by target site transcription. The implications of our observations on current models of transcription-stimulated recombination are discussed.
我们评估了靶位点转录对培养的人纤维肉瘤细胞中基因打靶的影响。构建了许多在其染色体DNA中含有质粒重组底物的细胞系。然后在存在和不存在刺激靶位点转录的试剂的情况下，在这些不同位点测量基因打靶频率。我们观察到RNA转录显著增强了基因打靶。转录刺激的基因打靶幅度从3倍到大于20倍不等。然而，当转录远离而非通过重组位点进行时，未观察到基因打靶增加。当与编码链或模板链互补的单链质粒载体用作重组底物时，也观察到了转录刺激的基因打靶。我们的结果表明，与其他形式的DNA重组一样，基因打靶可受到靶位点转录的刺激。讨论了我们的观察结果对当前转录刺激重组模型的影响。
