Both the CaT-Active (CaT-A) and CaT-Background (CaT-B) reagents contain lentiviruses that express RFP and GFP from separate transcripts, each driven by its own promoter. The RFP transcript is driven by the strong CMV promoter while the GFP gene is driven by the weak UbiC promoter (see Figure below).
The CaT-A lentivirus expresses an sgRNA targeting the UbiC promoter of the GFP gene. When transduced into cells, also expressing a functional dCas9-TA fusion protein (such as Cellecta’s dCas9-VPH), GFP expression increases in response to the sgRNA mediated dCas9-transactivation.
The CaT-B lentivirus also expresses an sgRNA but it is non-targeting so it cannot recruit the dCas9-transactivator and GFP expression is unaffected.
To assay cells for any dCas9-Activator activity, transduce two different populations of the same dCas9-TA expressing cells with the CaT-A and CaT-B viruses, respectively. GFP fluorescence in the cells transduced the CaT-A virus will be significantly increased, as compared with the CaT-B cells. RFP fluorescence in both populations is unaffected. The difference in the mean GFP fluorescence between the two populations (normalizing against mean RFP fluorescence) provides a quantitative measure of the activity of the dCas9-TA transactivator in the dCas9-TA cells.
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