Visualization of P-TEFb by Fluorescence Complementation
OwnerBoris Peterlin, Koh Fujinagainventors
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Abstract
Provided herein is a novel assay for the quantification of P-TEFb activation in living cells. The invention, in one embodiment, comprises cells which express P-TEFb and a P-TEFb-phosphorylated or P-TEFb-binding species, for example the C terminal domain of RNA polymerase II. Each of the P-TEFb and P-TEFb-phosphorylated or P-TEFb-binding species comprises a complementary signal moiety, for example complementary fragments of a fluorescent protein, such that when the P-TEFb interacts with the P-TEFb-phosphorylated or P-TEFb-binding species, the signal moieties are in sufficiently close proximity to generate a detectable signal.
Claims19
A cell which expresses
- a first polypeptide comprising a P-TEFb moiety, such P-TEFb moiety comprising a P-TEFb protein or biologically active fragment thereof, and a first signal moiety;
- and
- a second polypeptide comprising a P-TEFb target moiety and a second signal moiety;
- wherein when the first and second polypeptides are interacting, the first and second signal moieties are placed in proximity to each other, producing a detectable signal.
A method of determining the effect of a treatment on the release of P-TEFb in a living cell, wherein the cell expresses a first polypeptide comprising a P-TEFb moiety, such P-TEFb moiety comprising a P-TEFb protein or biologically active fragment thereof, and a first signal moiety, and the cell expresses a second polypeptide comprising a P-TEFb target moiety, a second signal moiety, and a nuclear localization signal, and wherein the first and second signal moieties, when in proximity to each other, produce a detectable signal, comprising the steps of
- subjecting the cell to a treatment;
- and
- subsequently observing the amount of signal in the treated cell;
- wherein, an elevated amount of signal observed in the treated cell, relative to control group cells, is indicative that the treatment increased the release of free P-TEFb.
Dependencies are inferred from the claim text.