pCDH-CuO-FLuc-P2A-GFP-EF1-CymR-T2A-Puro SparQ2 All-in-one Cloning and Expression Lentivector

Product Description

This all-in-one inducible vector belongs to the SparQ™2 Cumate Switch System. It is a gene regulatory system that gives researchers full control of the cells on when and how much a gene-of-interest (protein) can be expressed.

SparQ™2 is adding some of the key advantages over the previous generation and possesses several important elements for researchers to conduct gene modulation projects.

• P2A linker provides higher consistency for GFP expression
• Insert gene size up to 3 kb
• Robustness - increase expression up to 40-fold
• Adjustable - tune expression levels by titrating the amount of cumate
• Reversible - turn expression on, off, then on again
• Simple - the all-in-one format allows co-expression of CymR and your gene-of-interest
• Powerful - suitable for in vivo applications

How It Works

The regulatory mechanisms of the bacterial operons cmt and cym have been engineered to regulate gene expression in mammalian cells. In the repressor configuration, regulation is mediated by strong binding of the CymR repressor to the Cumate operator site (CuO), which is downstream of the CMV5 promoter. Addition of cumate (depicted as yellow circle in the graphic scheme below), a non-toxic small molecule inducer, relieves the repression and drives gene activation; in this case, fluorescent proteins (RFP and GFP). Subsequent removal of cumate from the growth media reverts the activation process, rendering gene switches to off mode.

Figure 1. Cumate Switch System

General Features of SBI’s SparQ Cumate Switch Systems

• CymR, a repressor that binds to cumate operator sequences in the absence of cumate
• CS, to clone-in your gene-of-interest
• Cumate inducible promoter with cumate operator sequences (CuO) upstream of the MCS, to inducibly control gene expression
• Variety of selection markers, to monitor the induction or select stable cell line
• CymR has a high binding affinity for cumate and, as more cumate is added, fewer CymR molecules bind to the CuO sequences in the promoter resulting in increased expression
• Exhibiting much lower background expression than similar systems

All-in-one Expression Lentivectors of the SparQ™2 System

• pCDH-CuO-MCS-P2A-GFP-EF1-CymR-T2A-Puro Plasmid
  
• pCDH-CuO-MCS-P2A-RFP-EF1-CymR-T2A-Puro Plasmid
• pCDH-CuO-MCS-P2A-GFP-EF1-CymR-T2A-Blast Plasmid
• pCDH-CuO-MCS-P2A-RFP-EF1-CymR-T2A-Blast Plasmid
• pCDH-CuO-MCS-P2A-GFP-EF1-CymR-T2A-Neo Plasmid
• pCDH-CuO-MCS-P2A-RFP-EF1-CymR-T2A-Neo Plasmid
• pCDH-CuO-RFP-P2A-GFP-EF1-CymR-T2A-Puro Plasmid
• pCDH-CuO-FLuc-P2A-GFP-EF1-CymR-T2A-Puro Plasmid

Supporting Data

Tight expression control, low background, and reversible with the SparQ™2 Cumate Switch System

Gene expression with the SparQ™2 Cumate Switch On/Off System

Figure 2. QM826B-1 was packaged into virus and infected HEK293 cells at MOI of 15. 3 days after infection, 1ug/ml puromycin was added into culture medium to establish stable cell line. Then QM826B-1 HEK293 stable cell line was treated with cumate at difference concentration. 3 days after cumate treatment, RFP and GFP expression was well induced. Cumate was then removed from the culture medium. After 3 days of cumate-free, RFP and GFP expression was barely noticeable.

Gene expression with the SparQ™2 Cumate Switch System is titratable and reversible

Figure 2. QM826B-1 was packaged into virus and infected HEK293 cells at MOI of 15. 3 days after infection, 1ug/ml puromycin was added into culture medium to establish stable cell line. Then QM826B-1 HEK293 stable cell line was treated with cumate at difference concentration. 3 days after cumate treatment, RFP and GFP expression was well induced. Cumate was then removed from the culture medium. After 3 days of cumate-free, RFP and GFP expression was barely noticeable.

Gene expression with the SparQ™2 Cumate Switch System is reversible and repeatable

Figure 4. QM828B-1 was packaged into virus and infected HEK293 cells at MOI of 15. 3 days after infection, 1µg/ml puromycin was added into culture medium to establish stable cell line. Then QM828B-1/HEK293 stable cell line was treated with cumate at 60µg/ul. 3 days after cumate treatment, luciferase expression was well induced. Then cumate was removed from the culture medium. After 3 days without cumate treatment, luciferase activity returned to the control level. Finally, when cumate was added back to the culture media, luciferase activity was once again induced.