Overview

An easy-to-produce non-integrating option for inducible gene expression

With virtually no limits on insert size (unlike AAV vectors) Enhanced Episomal Vectors (EEVs) are an excellent choice for non-integrating, non-viral gene expression. Because they replicate in synchrony with the host cell, they are stably inherited and can be used for long-lasting expression—up to several months both in vitro and in vivo—without modifying the host genome. Use SBI’s CuO-MCS EEV Vector (Cat.# EEV610A-1) for inducible expression of your gene-of-interest. The CuO promoter is a tightly-controlled inducible promoter which provides titratable gene expression in response to cumate.

SBI’s EEV System, which is an enhanced version of the Epstein-Barr Nuclear Antigen-1 (oriP-EBNA1) system, offers:

• High levels of expression
• Easy to clone formats
• No special plasmid production
• Nonviral, non-integrating technology
• Constitutive and inducible vector formats

Please note the following Licensing Restrictions for the EEV system:

For Academic and Non-Profit Institutions:

Researchers at academic and non-profit institutes are granted full access to purchasing the EEV cloning vectors and reporters. Full sequence information is provided upon proof of purchase of EEV vectors. Please contact SBI’s technical support at tech@systembio.com for sequence information.

For Commercial Customers:

For-profit and commercial customers can purchase the pre-made EEV reporters (EEV604A-1, EEV605A-1); however, due to licensing restrictions, cloning EEV vectors (e.g. EEV600A-1 and EEV610A-1) are not available for purchase directly. Instead, these are available through SBI’s custom EEV cloning and production services. SBI then provides the commercial customer with the desired amount of ready-to-use EEV custom construct DNA.

How It Works

Tightly-controlled, inducible gene expression

Get robust, titratable gene expression with low background using SBI’s cumate-inducible vectors. These vectors take advantage of CymR, a repressor that binds to cumate operator sequences (CuO) with high affinity in the absence of cumate, a non-toxic small molecule. Providing much lower background expression than similar systems, SparQ vectors can provide up to 32-fold induction of gene expression.

The cumate-inducible EEV Cloning and Expression vector CuO-MCS (Cat.# EEV610A-1) features a cumate-inducible promoter upstream of an MCS—cloning your gene-of-interest into the MCS will result in cumate-responsive expression of your gene-of-interest. The CuO-MCS plasmid also contains a constitutive EF1α promoter expressing the CymR repressor and a Puromycin selection cassette. The EEV610A-1 cumate switch inducible reporter is silent until the addition of cumate (Cat# QM150A-1).

• Robust—increase expression up to 32-fold
• Adjustable—tune expression levels by titrating the amount of cumate
• Reversible—turn expression on, then off, then on again
• Powerful—suitable for in vivo applications

Supporting Data

Sustained, inducible, and non-integrating gene expression in vitro and in vivo with EEV

Figure 1. The inducible EEV reporter CuO-GFP-T2A-Luciferase delivers robust gene expression in vitro over 72 days. A GFP-T2A-Luciferase cassette was cloned into CuO-MCS (note that this reporter construct is also available pre-built (CuO-GFP-T2A-Luciferase Cat.# EEV605A-1)) and transfected into HEK293 cells. Cumate was added to the medium daily at a final concentration of 300 µg/ml and cells imaged for GFP signal. GFP induction appeared after two days and expression detected for 72 days (2.5 months) after the original transfection, demonstrating just how long-lasting episomal expression delivered with EEV vectors can be.

Figure 2. The inducible EEV reporter CuO-GFP-T2A-Luciferase delivers robust gene expression in vivo. The Cumate inducible EEV reporter was also tested in mice. 5 µg of CuO-GFP-T2A-Luciferase plasmid (Cat.# EEV605A-1) was introduced into three test mice (n=3) via hydrodynamic tail vein injection (HDD) on Day 0. EEV expression was induced by IP injection of 1.5 mg of the water-soluble version of cumate (cat# QM150A-1). Mice were then imaged for luminescence activity through full body scans on Day 2. The high luciferase activity detected demonstrates the ability of the inducible EEV reporter to deliver robust gene expression upon induction.

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