A low-background Tet-On system based on post-transcriptional regulation using Csy4.
A low-background Tet-On system based on post-transcriptional regulation using Csy4.
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基于 Csy4 转录后调控的低背景 Tet-On 系统
DOI：
10.1371/journal.pone.0244732
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发表时间：
2020
期刊：
影响因子：
3.7
通讯作者：
Zhu Z
中科院分区：
文献类型：
--
作者：
Zhou Y;Lei C;Zhu Z
四环素调控系统由于其严格的调控和高的诱导率，被广泛用于真核生物中诱导目的基因的表达。然而，在某些情况下，其相关的背景表达可能是有问题的，如在高毒性蛋白质的表达中。我们发现，当使用Tet-On 3G系统驱动kid毒素基因在sf 9昆虫细胞中表达时，比在不存在诱导剂多西环素的情况下使用空载体时杀死更高百分比的细胞，从而表明该诱导型表达系统的泄漏表达。此外，我们发现四环素控制的转录沉默子（tTS）不能有效地降低sf 9细胞中Tet-On 3G系统的背景表达。然而，发现Csy 4（CRISPR家族中具有序列特异性核酸内切酶活性的Cas9同源蛋白）在减少Tet-On 3G系统相关的背景表达方面是有效的，尽管最大诱导表达伴随减少。然而，我们发现，通过结合TRE-controlled反义csy 4与WSSVie 1（Δ23）启动子驱动的正义csy 4对系统进行修饰显著降低了Tet-On 3G系统的泄漏表达，并且诱导水平高于最初获得的水平。这种优化的Tet-On 3G系统可以显著减少在未诱导条件下归因于Kid背景表达的细胞死亡。因此，我们开发了一种新的低背景诱导型表达系统，用于昆虫细胞，并可能在其他生物体，包括哺乳动物的基础上使用Csy 4的转录后调控。
On account of its stringent regulation and high rate of induction, the tetracycline regulatory system is used extensively for inducing target gene expression in eukaryotes. However, under certain circumstances, its associated background expression can be problematic, as in the expression of highly toxic proteins. We found that when using the Tet-On 3G system to drive expression of the kid toxin gene in sf9 insect cells, a higher percentage of cells were killed than when using an empty vector in the absence of the induction agent doxycycline, thereby indicating the leaky expression of this inducible expression system. Moreover, we found that the tetracycline-controlled transcriptional silencer (tTS) does not effectively reduce the background expression of the Tet-On 3G system in sf9 cells. However, Csy4, a Cas9 homologous protein in the CRISPR family with sequence-specific endonuclease activity, was found to be effective in reducing the Tet-On 3G system-associated background expression, although there was a concomitant reduction in the maximum induced expression. Nevertheless, we found that modification of the system via incorporation of TRE-controlled anti-sense csy4 in combination with a WSSVie1 (Δ23) promotor-driven sense csy4 significantly reduced the leaky expression of the Tet-On 3G system, and that the level of induction was higher than that initially obtained. This optimized Tet-On 3G system can significantly reduce cell death attributed to the background expression of Kid under uninduced conditions. Therefore, we developed a novel low-background inducible expression system for use in insect cells and potentially in other organisms including mammals based on post-transcriptional regulation using Csy4.
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影响因子：
--
作者：
Park, Da-Young;Lee, Jung-Hwan;Ko, Kisung
通讯作者：
Ko, Kisung
影响因子：
7.2
作者：
WEINMANN, P;GOSSEN, M;GATZ, C
通讯作者：
GATZ, C
影响因子：
4.1
作者：
Wu, TY;Liono, L;Chao, YC
通讯作者：
Chao, YC
影响因子：
14.9
作者：
Forster, K;Helbl, V;Hillen, W
通讯作者：
Hillen, W
DOI：
10.1126/science.1192272
发表时间：
2010-09-10
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Haurwitz RE;Jinek M;Wiedenheft B;Zhou K;Doudna JA
通讯作者：
Doudna JA