Genomic cis-acting Sequences Improve Expression and Establishment of a Nonviral Vector.-猫眼课题宝

DOI：

10.1038/mtna.2013.47

发表时间：

2013-09-03

期刊：

Molecular therapy. Nucleic acids

影响因子：

通讯作者：

中科院分区：

其他

文献类型：

作者：

来源链接：

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3759742/?report=classic

载体pEPI是第一个非病毒和附加型复制载体。其功能元件是与染色体支架/基质附着区（S/MAR）连接的表达单元。该载体在各种细胞系中以低拷贝数自主复制，在没有选择的情况下在数百代中是有丝分裂稳定的，并且成功地用于高效产生转基因猪。由于假设载体的建立是随机事件并且强烈地依赖于其在转染后到达的核区室，因此鉴定引导DNA序列进入某些核区室的基因组序列是非常有意义的。在这里，我们将基因组顺式作用序列插入到pEPI中，并检查它们对转基因表达、长期稳定性和载体建立的影响。我们证明，一个普遍存在的染色质开放元件（UCOE）介导增强转基因表达，而绝缘子序列（cHS 4）增加建立效率，大概是通过一个额外的相互作用与核基质。因此，除了作为一个有前途的替代目前使用的病毒载体在基因治疗方法，pEPI也可以作为一种工具，研究核区室化;识别基因组顺式作用序列，参与核组织将有助于我们理解之间的相互作用转基因表达，质粒的建立，和核结构。

The vector pEPI was the first nonviral and episomally replicating vector. Its functional element is an expression unit linked to a chromosomal scaffold/matrix attached region (S/MAR). The vector replicates autonomously with low copy number in various cell lines, is mitotically stable in the absence of selection over hundreds of generations, and was successfully used for the efficient generation of genetically modified pigs. Since it is assumed that establishment of the vector is a stochastic event and strongly depends on the nuclear compartment it reaches after transfection, it is of great interest to identify genomic sequences that guide DNA sequences into certain nuclear compartments. Here we inserted genomic cis-acting sequences into pEPI and examined their impact on transgene expression, long-term stability, and vector establishment. We demonstrated that a ubiquitous chromatin-opening element (UCOE) mediated enhanced transgene expression, while an insulator sequence (cHS4) increased establishment efficiency, presumably via an additional interaction with the nuclear matrix. Thus, besides being a promising alternative to currently used viral vectors in gene therapeutic approaches, pEPI may also serve as a tool to study nuclear compartmentalization; identification of genomic cis-acting sequences that are involved in nuclear organization will contribute to our understanding of the interplay between transgene expression, plasmid establishment, and nuclear architecture.