Hejnar J, Hájková P, Plachy J, Elleder D, Stepanets V, Svoboda J
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, CZ 16637 Prague 6, Czech Republic.
Proc Natl Acad Sci U S A. 2001 Jan 16;98(2):565-9. doi: 10.1073/pnas.98.2.565.
CpG islands are important in the protection of adjacent housekeeping genes from de novo DNA methylation and for keeping them in a transcriptionally active state. However, little is known about their capacity to protect heterologous genes and assure position-independent transcription of adjacent transgenes or retroviral vectors. To tackle this question, we have used the mouse aprt CpG island to flank a Rous sarcoma virus (RSV)-derived reporter vector and followed the transcriptional activity of integrated vectors. RSV is an avian retrovirus which does not replicate in mammalian cells because of several blocks at all levels of the replication cycle. Here we show that our RSV-derived reporter proviruses linked to the mouse aprt gene CpG island remain undermethylated and keep their transcriptional activity after stable transfection into both avian and nonpermissive mammalian cells. This effect is most likely caused by the protection from de novo methylation provided by the CpG island and not by enhancement of the promoter strength. Our results are consistent with previous finding of CpG islands in proximity to active but not inactive proviruses and support further investigation of the protection of the gene transfer vectors from DNA methylation.
CpG岛对于保护邻近的管家基因免受从头DNA甲基化影响并使其保持转录活性状态至关重要。然而，关于它们保护异源基因以及确保邻近转基因或逆转录病毒载体的位置独立转录的能力，我们却知之甚少。为了解决这个问题，我们使用小鼠aprt CpG岛侧翼一个源自劳氏肉瘤病毒（RSV）的报告载体，并追踪整合载体的转录活性。RSV是一种禽逆转录病毒，由于在复制周期的各个层面存在多个障碍，它不能在哺乳动物细胞中复制。在这里我们表明，与小鼠aprt基因CpG岛相连的源自RSV的报告前病毒在稳定转染到禽类和非允许性哺乳动物细胞后仍保持低甲基化并维持其转录活性。这种效应很可能是由CpG岛提供的免受从头甲基化的保护引起的，而不是由启动子强度的增强导致的。我们的结果与之前在活跃而非不活跃前病毒附近发现CpG岛的结果一致，并支持进一步研究基因转移载体免受DNA甲基化的保护作用。
