pTiGer Cloning Plasmids

pTiGer-mcs: plasmids for Tet-inducible Gene expression or repression

Tetracycline-inducible pTiGer-mcs plasmid

InvivoGen’s pTiGer-mcs plasmid family features a Tetracycline (Tet)-inducible expression cassette containing the strong composite CMV-EF1 promoter modified to integrate a tetracycline response element (TRE), a convenient multiple cloning site (mcs), and an efficient terminator of transcription. Each plasmid carries an antibiotic resistance marker for selection in both mammalian cells and bacteria.
Our pTiGer-mcs plasmids are functionally validated by transfecting the secreted embryonic alkaline phosphatase (SEAP) into tetracycline repressor-expressing cells (see below).

— pTiGer2-mcs is selectable using Zeocin^®

— pTiGer3-mcs is selectable using Hygromycin

— pTiGer4-mcs is selectable using Puromycin

Induce the GOI expression in just three steps

Step 1: The GOI is cloned into a pTiGer plasmid. * Save time and effort with our custom cloning service (see below).

Step 2: The pTiGer-GOI plasmid is transiently or stably transfected into HEK-RepTor™ or A549-RepTor™ cells. These cells express the tetracycline repressor (TetR) which binds to the TRE within the plasmid, repressing GOI expression [1].

Step 3: The addition of doxycycline, a synthetic derivative of the tetracycline antibiotic, leads to TetR release and GOI transcription.

Of note, the composite Tet-inducible CMV-EF1-TRE promoter in the pTiGer plasmids confers strong expression and full induction of the GOI in the presence of 1 ng/ml of doxycycline. This low antibiotic dose ensures minimal toxicity or metabolic changes [2].

Save time and effort with our full custom cloning service

InvivoGen helps you expedite the cloning of your GOI into the pTiGer plasmid of your choice. Depending on your GOI, two service options are provided:

Direct cloning: Find your GOI in our extensive collection of human or mouse genes.

Gene synthesis and cloning: Your GOI is not listed yet in our gene collection. Our expert team evaluates the feasibility based on the sequence you provide, then proceeds to the gene synthesis and cloning into the pTiGer plasmid.

This cost-effective service guarantees rapid processing and confirmation of the plasmid construct using restriction analysis and full-length open reading frame (ORF) sequencing. After purification by ion exchange chromatography, predominant supercoiled conformation is verified by electrophoresis. Your customized pTiGer plasmid is available within 3-5 weeks. Contact us for more information.

Choose among pTiGer-reporter vectors for controls

Invivogen offers pTiGer control plasmids featuring a tetracycline-inducible reporter gene, either SEAP (secreted embryonic alkaline phosphatase), Lucia luciferase, or eGFP (enhanced green fluorescent protein), and a choice of selectable markers.

References:

1. Hillen, W., Wissmann, A. (1989). Tet repressor-tet operator interaction. Protein-Nucleic Acid Interaction. DOI: 10.1007/978-1-349-09871-2_7.
2. Moullan, N., et al., (2015). Tetracyclines disturb mitochondrial function across eukaryotic models: a call for caution in biomedical research. Cell Rep. 10(10):1681-91. 

Specifications

No transcriptional interference: the two expression cassettes for the gene of interest and the selection marker are oriented in convergent directions.

Strong and inducible expression of the gene of interest: CMV-EF1-TRE composite promoter.

Convenient Multiple Cloning Site: AgeI, BstEII, NcoI, BamHI, Acc65I, XbaI, NsiI, EcoRV, and NheI.

Features:

• pTiGer2-mcs is selectable with Zeocin® in mammalian cells and E.coli
• pTiGer3-mcs is selectable with Hygromycin in mammalian cells and E.coli
• pTiGer4-mcs is selectable with Puromycin in mammalian cells and E.coli

Contents

Note: Each plasmid is sold separately.

pTiGer2-mcs

• 20 µg of lyophilized DNA
• 1 ml of Zeocin^® (100 mg/ml)

pTiGer3-mcs

• 20 µg of lyophilized DNA
• 1 ml of Hygromycin (100 mg/ml)

pTiGer4-mcs

• 20 µg of lyophilized DNA
• 1 ml of Puromycin (10 mg/ml)

 The product is shipped at room temperature.

Details

TiGer Tet-on assay principle

InvivoGen's TiGer Tet-on system relies on the combined use of two plasmids; the first expressing an optimized TetR construct, and the second, pTiGer, a GOI under the control of an engineered tetracycline operator (tetO)-containing promoter. It features the strong composite CMV-EF1 promoter modified to integrate a tetracycline response element (TRE).

The TetR plasmid was stably transfected into the RepTor™ cell lines, to save you time and effort. Upon transfection of the RepTor™ cells with a pTiGer-GOI plasmid, TetR binds to TRE and blocks the transcription of the GOI. Addition of doxycycline, a synthetic analog of tetracycline, leads to the release of TetR from the tetO sequences, resulting in the derepression of the CMV-EF1-TRE promoter and transcription of the GOI.

The TiGer Tet-on system has been engineered to guarantee maximal repression of the GOI, ensuring minimal leakage of GOI expression in the absence of tetracycline/doxycycline, and strong GOI expression in the presence of the antibiotic. This key feature confers the TiGer Tet-on system with the advantage of working with cytotoxic genes (e.g. proteases, pore-forming proteins, gain-of-function variants).

TiGer Tet-on assay system

Citations