دورية أكاديمية
A low-background Tet-On system based on post-transcriptional regulation using Csy4.
| العنوان: | A low-background Tet-On system based on post-transcriptional regulation using Csy4. |
|---|---|
| المؤلفون: | Yicheng Zhou, Chaoliang Lei, Zhihui Zhu |
| المصدر: | PLoS ONE, Vol 15, Iss 12, p e0244732 (2020) |
| بيانات النشر: | Public Library of Science (PLoS), 2020. |
| سنة النشر: | 2020 |
| المجموعة: | LCC:Medicine LCC:Science |
| مصطلحات موضوعية: | Medicine, Science |
| الوصف: | 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. |
| نوع الوثيقة: | article |
| وصف الملف: | electronic resource |
| اللغة: | English |
| تدمد: | 1932-6203 |
| Relation: | https://doaj.org/toc/1932-6203 |
| DOI: | 10.1371/journal.pone.0244732 |
| URL الوصول: | https://doaj.org/article/8db38f24bf7b44a5a10d3b1d8bade8e6 |
| رقم الأكسشن: | edsdoj.8db38f24bf7b44a5a10d3b1d8bade8e6 |
| قاعدة البيانات: | Directory of Open Access Journals |
Full Text Finder | تدمد: | 19326203 |
|---|---|
| DOI: | 10.1371/journal.pone.0244732 |