A Self-Replicating Linear DNA.,ACS Synthetic Biology

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A Self-Replicating Linear DNA.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-03-26 , DOI: 10.1021/acssynbio.9b00478
Pei Sheng Liew ₁ , Tze Hao Tan ₂ , Yin Cheng Wong ₁ , Edmund Ui Hang Sim ₃ , Choon Weng Lee ₄ , Kumaran Narayanan ₁

Affiliation

TelN and tos are a unique DNA linearization unit isolated from bacteriophage N15. While being transferable, the TelN cleaving-rejoining activities remained stable to function on tos in both bacterial and mammalian environments. However, TelN contribution in linear plasmid replication in mammalian cells remains unknown. Herein, we investigated the association of TelN in linear tos-containing DNA (tos-DNA) replication in mammalian cells. Additionally, the mammalian origin of replication (ori) that is well-known to initiate the replication event of plasmid vectors was also studied. In doing so, we identified that both TelN and mammalian initiation sites were essential for the replication of linear tos-DNA, determined by using methylation sensitive DpnI/MboI digestion and polymerase chain reaction (PCR) amplification approaches. Furthermore, we engineered the linear tos-DNA to be able to retain in mammalian cells using S/MAR technology. The resulting S/MAR containing tos-DNA was robust for at least 15 days, with (1) continuous tos-DNA replication, (2) correct splicing of gene transcripts, and (3) stable exogenous gene expression that was statistically comparable to the endogenous gene expression level. Understanding the activities of TelN and tos in mammalian cells can potentially provide insights for adapting this simple DNA linearization unit in developing novel genetic engineering tools, especially to the eukaryotic telomere/telomerase study.

中文翻译：

自复制线性DNA。

TelN和tos是从噬菌体N15中分离出的独特的DNA线性化单位。尽管是可转移的，但TelN裂解-重结合活性在细菌和哺乳动物环境中均能稳定发挥作用。然而，TelN在哺乳动物细胞中线性质粒复制中的贡献仍然未知。在这里，我们研究了哺乳动物细胞中TelN与线性tos含DNA（tos-DNA）复制的关联。另外，还研究了众所周知的启动质粒载体复制事件的哺乳动物复制起点（ori）。通过这样做，我们确定TelN和哺乳动物的起始位点对于线性tos-DNA的复制都是必不可少的，这是通过使用甲基化敏感性DpnI / MboI消化和聚合酶链反应（PCR）扩增方法确定的。此外，我们设计了线性tos-DNA，使其能够使用S / MAR技术保留在哺乳动物细胞中。所得的含tos-DNA的S / MAR至少在15天内很强，并且（1）tos-DNA连续复制，（2）基因转录物的正确剪接和（3）稳定的外源基因表达在统计学上可与内源基因表达水平。了解TelN和tos在哺乳动物细胞中的活性可能为开发这种简单的DNA线性化单元提供新颖的基因工程工具，尤其是对真核端粒/端粒酶研究的适应性见解。（3）稳定的外源基因表达，在统计学上与内源基因表达水平相当。了解TelN和tos在哺乳动物细胞中的活性可能为开发这种简单的DNA线性化单元提供新颖的基因工程工具，尤其是对真核端粒/端粒酶研究的适应性见解。（3）稳定的外源基因表达，在统计学上与内源基因表达水平相当。了解TelN和tos在哺乳动物细胞中的活性可能为开发这种简单的DNA线性化单元提供新颖的基因工程工具，尤其是对真核端粒/端粒酶研究的适应性见解。

更新日期：2020-04-23

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