Institute of Medical Microbiology and Hygiene, Molecular Microbiology & Gene Therapy Unit, University of Regensburg, Regensburg, Germany.
Nucleic Acids Res. 2010 Jul;38(12):3891-908. doi: 10.1093/nar/gkq115. Epub 2010 Mar 4.
The development of vaccine components or recombinant therapeutics critically depends on sustained expression of the corresponding transgene. This study aimed to determine the contribution of intragenic CpG content to expression efficiency in transiently and stably transfected mammalian cells. Based upon a humanized version of green fluorescent protein (GFP) containing 60 CpGs within its coding sequence, a CpG-depleted variant of the GFP reporter was established by carefully modulating the codon usage. Interestingly, GFP reporter activity and detectable protein amounts in stably transfected CHO and 293 cells were significantly decreased upon CpG depletion and independent from promoter usage (CMV, EF1 alpha). The reduction in protein expression associated with CpG depletion was likewise observed for other unrelated reporter genes and was clearly reflected by a decline in mRNA copy numbers rather than translational efficiency. Moreover, decreased mRNA levels were neither due to nuclear export restrictions nor alternative splicing or mRNA instability. Rather, the intragenic CpG content influenced de novo transcriptional activity thus implying a common transcription-based mechanism of gene regulation via CpGs. Increased high CpG transcription correlated with changed nucleosomal positions in vitro albeit histone density at the two genes did not change in vivo as monitored by ChIP.
疫苗成分或重组治疗剂的开发严重依赖于相应转基因的持续表达。本研究旨在确定内含子 CpG 含量对瞬时和稳定转染哺乳动物细胞中表达效率的贡献。基于包含其编码序列内 60 个 CpG 的人源化绿色荧光蛋白 (GFP)，通过仔细调节密码子使用，建立了 GFP 报告基因的 CpG 耗尽变体。有趣的是，GFP 报告基因活性和可检测的蛋白量在稳定转染的 CHO 和 293 细胞中显著降低，而与启动子使用（CMV、EF1 alpha）无关。与 CpG 耗竭相关的蛋白表达减少也观察到其他不相关的报告基因，并且mRNA 拷贝数的下降而不是翻译效率明显反映出来。此外，mRNA 水平的降低既不是由于核输出限制，也不是由于选择性剪接或 mRNA 不稳定性。相反，内含子 CpG 含量影响从头转录活性，从而暗示 CpG 通过共同的基于转录的基因调控机制。体外高 CpG 转录与核小体位置的改变相关，尽管体内通过 ChIP 监测到两个基因的组蛋白密度没有改变。
