Anastasis biosensor
The present invention relates to the field of anastasis, i.e., the process of reversal of apoptosis. More specifically, the present invention provides methods and compositions useful for studying anastasis. In one embodiment, the present invention provides an in vivo biosensor comprising (a) a transcription factor complex comprising the Gal4 transcription factor linked to an enzyme cleavable linker, wherein the transcription factor complex is tethered to the plasma membrane via a transmembrane domain; and (b) a reporter system comprising (1) a first nucleic acid encoding flippase operably linked to the upstream activating sequence that binds Gal4; and (2) a second nucleic acid comprising an FRT-flanked stop codon cassette separating a constitutive promoter and a fluorescent protein open reading frame.
1. A method for identifying cells that survive apoptosis via anastasis comprising the steps of:
a. transiently inducing apoptosis in a cell comprising an anastasis biosensor, wherein the anastasis biosensor comprises:
(i) a transcription factor complex comprising a Gal4 transcription factor linked to a transmembrane domain via a caspase enzyme cleavable linker, wherein the transcription factor complex is tethered to the plasma membrane of the cell in which the in vivo anastasis biosensor is expressed, and wherein the caspase enzyme is specifically expressed during apoptosis, and
(ii) a reporter system comprising (1) a first nucleic acid encoding flippase operably linked to the upstream activating sequence that binds Gal4; and (2) a second nucleic acid comprising a flippase recognition target (FRT)-flanked stop codon cassette separating a constitutive promoter and a fluorescent protein open reading frame,
b. detecting fluorescent protein expression, wherein a cell expressing the in vivo anastasis biosensor that survives apoptosis via anastasis expresses the fluorescent protein.
2. The method of claim 1 , wherein the fluorescent protein comprises green fluorescent protein, red fluorescent protein, or yellow fluorescent protein.