pGreenFire 2.0 TCF/LEF reporter plasmid (pGF2-TCF/LEF-rFluc-T2A-GFP-mPGK-Puro)

SBI has upgraded our popular pGreenFire signaling pathway reporter lentivectors with a design that leads to more reliable generation of stable cell lines. We’ve also swapped in the red firefly luciferase reporter (rFLuc), which opens up the possibility of performing a dual-spectral luciferase assay and delivers greater sensitivity for in vivo applications than conventional luciferase.

What makes our next-gen pGreenFire 2.0 vectors even better than other TRE reporter vectors is the smart design, which adds in a constitutive selection cassette for stable cell line generation while minimizing interference with the upstream TRE. By using a weak/moderate mPGK promoter to drive the antibiotic selection marker (puromycin resistance) and carefully arranging the conditional reporter genes, the selection marker is reliably expressed without compromising conditional expression of rFLuc and GFP.

With the pGreenFire 2.0 TCF/LEF Reporter Lentivector, Virus, and clonally-derived Stable Cell Line (pGF2-TCF/LEF-rFluc-T2A-GFP-mPGK-Puro), the core reporter functionality is similar to the original pGreenFire lentivector—TCF/LEF transcriptional response elements (TREs) are placed upstream of a minimal CMV promoter (mCMV) which together drive co-expression of rFLuc and GFP in response to TCF/LEF activity. The result is the ability to quantitatively measure NF-κB activity using both fluorescence and luciferase activity.

As with our original pGreenFire1 vectors, all pGreenFire 2.0 lentivectors leverage our reliable lentivector technology and save you time with pre-built signal transduction pathway reporters that come as ready-to-transduce pre-packaged lentivirus and plasmid that can be transfected into the lentivirus producing system of your choice*.

• Sort responsive cells with GFP
• Measure activity with red firefly luciferase
• Leverage SBI’s highly-regarded lentivectors
• Create stable signaling pathway reporter cell lines
• Introduce reporters into difficult-to-transfect cell types, including primary and non-dividing mammalian cell lines

Supporting Data

See our pGreenFire 2.0 transcriptional response element reporters in action

 

Figure 1. The pGreenFire 2.0 TCF/LEF Lentivector efficiently and quantitatively reports on TCF/LEF activity in 293T cells. Relative luciferase activity (A) and GFP activity (B) both increase in response to LiCl, a TCF/LEF inducer. Induction is specific to the TCF/LEF transcription response elements as the pGreenFire 2.0 mCMV Negative Control displays very little luciferase activity regardless of the presence of inducer.

Figure 2. The pGreenFire 2.0 TCF/LEF reporter cassette efficiently and quantitatively reports on TCF/LEF activity in the pGreenFire 2.0 TCF/LEF clonal 293T reporter cell line. Luciferase activity (A) and GFP intensity (B) both increase in response to LiCl.