Tet Regulatory Protein Expression Cell Lines
VectorBuilder offers Tet regulatory protein-expressing cell lines derived from single clones, providing consistency and efficiency for constructing inducible gene expression systems. Your target gene, driven by an inducible promoter such as TRE or TRE3G, can be introduced into these premade cells using viruses or plasmids, enabling flexible experimental design and reliable induction of gene expression.
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Shipping and storage
Cells are shipped on dry ice (in 10% DMSO-containing culture medium). Upon receipt, please immediately transfer the cryovials to the liquid nitrogen vapor phase for long-term storage. You can also store the cells at -80°C for short-term storage (≤1 week).
Note: For any premade stable cell lines purchased from VectorBuilder, customers agree not to resell them in any form, including in their original form, as copies, as aliquots, or in repackaged form, to any third party, unless stipulated differently in a separate agreement with VectorBuilder.
Technical Information
Figure 1. Simplified workflow for using VectorBuilder’s tTS/rtTA-expressing stable cell lines.
To achieve Tet-inducible gene expression, simply transfect or transduce VectorBuilder’s premade tTS/rtTA expression cells with a TRE-driven GOI expression plasmid or virus. Inducible expression of the GOI can be assessed using appropriate assays such as RT-qPCR, Western Blot, or fluorescence imaging, following induction with the proper concentration of doxycycline or tetracycline.
For more detailed information, please refer to our Inducible Gene Expression Solutions and User Instructions for Tet-inducible Gene Expression Vector Systems. To design a customized plasmid for inducible GOI expression for use in our Tet regulatory protein expression cell lines, please visit our Vector Design Studio or contact us for assistance.
Experimental Validation
Figure 2. Validation of tTS/rtTA-expressing HEK293 cells. Cells were transduced with TRE-driven EGFP-expressing lentivirus, followed by antibiotic selection. EGFP expression was assessed by fluorescence imaging 48 hours after induction with 2 µg/ml doxycycline (Dox) (+ Dox) or in the absence of Dox (- Dox). Magnification: 100×.
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FAQ
Are the premade tool cell lines derived from mixed pools or single clones?
All of VectorBuilder’s premade stable cell lines are derived from single clones to ensure consistency and reproducibility. Our single clone stable cell lines undergo stringent QC and functional validation to provide you with full confidence in downstream applications.
In the Tet Regulatory Protein Expression Cell Line, which inducible promoter should I use to drive my gene of interest?
The appropriate inducible promoter depends on the specific Tet regulatory protein expressed in your cell line. If your cell line expresses tTS/rtTA (2nd generation), the gene of interest should be driven by the TRE promoter. If your cells express Tet3G (3rd generation), the gene should be placed under the TRE3G promoter, which is specifically activated by Tet3G in the presence of tetracycline or its analogs (e.g., doxycycline). Using the correct promoter ensures proper inducible control of gene expression in your Tet-based system. For more details, please visit our Mammalian Inducible Promoters page. To design an appropriate inducible expression vector, use our Vector Design Studio or contact us for assistance.
Featured Citations
A lysine-rich cluster in the N-BAR domain of ARF GTPase-activating protein ASAP1 is necessary for binding and bundling actin filaments
Anjelika Gasilina, et al.
J Biol Chem, 2022, doi: 10.1016/j.jbc.2022.101700
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Abstract: Actin filament maintenance is critical for both normal cell homeostasis and events associated with malignant transformation. The ADP-ribosylation factor GTPase-activating protein ASAP1 regulates the dynamics of filamentous actin-based structures, including stress fibers, focal adhesions, and circular dorsal ruffles. Here, we have examined the molecular basis for ASAP1 association with actin. Using a combination of structural modeling, mutagenesis, and in vitro and cell-based assays, we identify a putative-binding interface between the N-Bin-Amphiphysin-Rvs (BAR) domain of ASAP1 and actin filaments. We found that neutralization of charges and charge reversal at positions 75, 76, and 79 of ASAP1 reduced the binding of ASAP1 BAR-pleckstrin homology tandem to actin filaments and abrogated actin bundle formation in vitro. In addition, overexpression of actin-binding defective ASAP1 BAR-pleckstrin homology [K75, K76, K79] mutants prevented cellular actin remodeling in U2OS cells. Exogenous expression of [K75E, K76E, K79E] mutant of full-length ASAP1 did not rescue the reduction of cellular actin fibers consequent to knockdown of endogenous ASAP1. Taken together, our results support the hypothesis that the lysine-rich cluster in the N-BAR domain of ASAP1 is important for regulating actin filament organization.
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The KU-PARP14 axis differentially regulates DNA resection at stalled replication forks by MRE11 and EXO1
Ashna Dhoonmoon, Claudia M. Nicolae & George-Lucian Moldovan.
Nature Communications, 2022, doi: 10.1038/s41467-022-32756-5
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Abstract: Suppression of nascent DNA degradation has emerged as an essential role of the BRCA pathway in genome protection.In BRCA-deficient cells, the MRE11 nuclease is responsible for both resection of reversed replication forks, and accumulation of single stranded DNA gaps behind forks. Here, we show that the mono-ADP-ribosyltransferase PARP14 is a critical co-factor of MRE11. PARP14 is recruited to nascent DNA upon replication stress in BRCA-deficient cells, and through its catalytic activity, mediates the engagement of MRE11. Loss or inhibition of PARP14 suppresses MRE11-mediated fork degradation and gap accumulation, and promotes genome stability and chemoresistance of BRCA-deficient cells. Moreover, we show that the KU complex binds reversed forks and protects them against EXO1-catalyzed degradation. KU recruits the PARP14-MRE11 complex, which initiates partial resection to release KU and allow long-range resection by EXO1. Our work identifies a multistep process of nascent DNA processing at stalled replication forks in BRCA-deficient cells.
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