A system for creating stable cell lines that express a gene of interest from a bidirectional and regulatable herpes simplex virus type 1 promoter.
A system for creating stable cell lines that express a gene of interest from a bidirectional and regulatable herpes simplex virus type 1 promoter.
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DOI：
10.1371/journal.pone.0122253
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Wilber A
中科院分区：
文献类型：
--
作者：
Chambers CB;Halford WP;Geltz J;Villamizar O;Gross J;Embalabala A;Gershburg E;Wilber A
由于三个主要因素，用于研究目的基因生物学功能的表达系统可能具有有限的实用性：i)弱的或异质性的基因表达；ii)基因表达控制不良；iii)稳定整合和持续表达的低效率。我们设想，理想的系统应该受到严格的控制，并结合有效地创建和鉴定稳定的细胞系的能力。在这里，我们描述了一个基于双向单纯疱疹病毒1型启动子的系统，该启动子对VP16反式激活子自然产生反应，并被修改以允许一侧四环素调节的转录，同时保持另一侧的构成活性。将该元件引入睡美人转座子中，得到了一种新型的双向系统，具有高效稳定整合的能力。使用这个系统，我们创建了稳定的细胞系，在这些细胞系中，通过一种新的组合-多西环素敏感的去抑制和VP16介导的序列特异性诱导，目的基因的表达在广泛的水平上受到严密和统一的控制。该系统的独特特性解决了当前方法的主要局限性，并为研究哺乳动物模型中基因剂量的影响提供了一种极好的策略。
Expression systems used to study the biological function of a gene of interest can have limited utility due to three major factors: i) weak or heterogeneous gene expression; ii) poorly controlled gene expression; and iii) low efficiencies of stable integration and persistent expression. We envisioned that the ideal system should be tightly controlled and coupled with the ability to efficiently create and identify stable cell lines. Herein, we describe a system based upon a bidirectional Herpes simplex virus type 1 promoter that is naturally responsive to the VP16 transactivator and modified to permit tetracycline-regulated transcription on one side while maintaining constitutive activity on the other side. Incorporation of this element into the Sleeping Beauty transposon resulted in a novel bidirectional system with the capacity for high-efficiency stable integration. Using this system, we created stable cell lines in which expression of a gene of interest was tightly and uniformly controlled across a broad range of levels via a novel combination of doxycycline-sensitive de-repression and VP16-mediated sequence-specific induction. The unique characteristics of this system address major limitations of current methods and provide an excellent strategy to investigate the effects of gene dosing in mammalian models.
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影响因子：
5.4
作者：
Halford, WP;Kemp, CD;Schaffer, PA
通讯作者：
Schaffer, PA
影响因子：
5.1
作者：
Curtin, J. A.;Dane, A. P.;Ginn, S. L.
通讯作者：
Ginn, S. L.
DOI：
10.1073/pnas.93.11.5185
发表时间：
1996-05-28
影响因子：
11.1
作者：
Hofmann, A;Nolan, GP;Blau, HM
通讯作者：
Blau, HM
影响因子：
64.5
作者：
Akhtar, Waseem;de Jong, Johann;van Steensel, Bas
通讯作者：
van Steensel, Bas
影响因子：
64.5
作者：
Ivics, Z;Hackett, PB;Izsvak, Z
通讯作者：
Izsvak, Z