利用携带合成启动子的逆转录病毒载体调节异位基因表达水平。

Systems and Synthetic Biology Pub Date : 2011-12-01 Epub Date: 2011-11-20 DOI:10.1007/s11693-011-9089-0
Joshua P Ferreira, Ryan W S Peacock, Ingrid E B Lawhorn, Clifford L Wang
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引用次数: 31

摘要

未标记:人巨细胞病毒和延伸因子1α启动子是哺乳动物表达载体常用的组成启动子。这些启动子通常在许多类型的细胞和组织中产生高水平的表达。为了生成能够产生一系列低、中、高表达水平的合成启动子库,对这些启动子的TATA和CAAT盒元件进行了突变。其他启动子变异也通过随机诱变产生。利用整合在基因组单个位点的质粒载体进行评估显示,这些不同的合成启动子的表达水平能够跨越40倍的范围。逆转录病毒载体上装配了合成的启动子,并评估了它们通过质粒整合重现分级表达的能力。具有自我失活的长末端重复序列的载体既不能复制全范围的表达水平,也不能产生稳定的表达。使用第二种载体设计,不同的合成启动子能够在不同细胞系的广泛表达水平上稳定表达。电子补充资料:本文的在线版本(doi:10.1007/s11693-011-9089-0)包含补充资料,可供授权用户使用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters.

Unlabelled: The human cytomegalovirus and elongation factor 1α promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines.

Electronic supplementary material: The online version of this article (doi:10.1007/s11693-011-9089-0) contains supplementary material, which is available to authorized users.

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