Anastasis biosensor

Abstract

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 . 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.   
     
     
         2 . The biosensor of  claim 1 , wherein the enzyme cleavable linker is cleaved by an enzyme specifically expressed during apoptosis. 
     
     
         3 . The biosensor of  claim 1 , wherein the enzyme is caspase. 
     
     
         4 . The biosensor of  claim 1 , wherein the fluorescent protein comprises green fluorescent protein, red fluorescent protein, or yellow fluorescent protein. 
     
     
         5 . A transgenic mammal comprising the biosensor of  claim 1 . 
     
     
         6 . A biosensor for studying anastasis comprising:
 a. a caspase-activatable transcription factor complex comprising Gal4 transcription factor linked to (1) a caspase-cleavable linker and (2) a transmembrane domain; and   b. a reporter system comprising a (1) first nucleic acid encoding flippase operably linked to the upstream activating sequence that binds Gal4; (2) a second nucleic acid comprising an FRT-flanked stop cassette separating a constitutive promoter and a fluorescent protein open reading frame.   
     
     
         7 . The biosensor of  claim 6 , the caspase-cleavable linker comprises the amino acid sequence DEVD. 
     
     
         8 . The biosensor of  claim 6 , wherein the constitutive promoter is the ubiquitin promoter. 
     
     
         9 . A biosensor comprising:
 a. a caspase-activatable transcription factor complex comprising Gal4 transcription factor linked to (1) a caspase-cleavable linker and (2) a transmembrane domain; and   b. the G-TRACE reporter system.   
     
     
         10 . A biosensor system comprising:
 a. a caspase-activatable recombinase complex comprising Cre recombinase protein linked to (1) a caspase-cleavable linker and (2) a transmembrane domain; and   b. a nucleic acid comprising a LoxP-flanked stop cassette separating a constitutive promoter and a fluorescent protein open reading frame.   
     
     
         11 . A biosensor system comprising:
 a. a site-specific recombinase tethered to the plasma membrane of a test cell, wherein the recombinase is linked to a transmembrane domain via an enzyme cleavable linker; and   b. a nucleic acid encoding a reporter gene operably linked to a promoter, wherein the recognition target sequence of the recombinase flanks a stop codon cassette located between the reporter gene and the promoter.   
     
     
         12 . The biosensor system of  claim 11 , wherein the site-specific recombinase is flippase and the recognition target sequence is FRT. 
     
     
         13 . The biosensor system of  claim 11 , wherein the reporter gene encodes a fluorescent protein. 
     
     
         14 . The biosensor system of  claim 11 , wherein the promoter is a constitutive promoter. 
     
     
         15 . The biosensor system of  claim 11 , wherein the enzyme is caspase. 
     
     
         16 . An in vivo biosensor comprising:
 a. a transcription factor complex comprising a 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 a site specific recombinase operably linked to the site specific sequence for the transcription factor; and (2) a second nucleic acid comprising a stop codon cassette flanked by site specific recombination sequences, wherein the stop codon cassette and flanking sequences separate a constitutive promoter and a fluorescent protein open reading frame.   
     
     
         17 . The biosensor of  claim 16 , wherein the enzyme cleavable linker is cleaved by an enzyme specifically expressed during apoptosis. 
     
     
         18 . The biosensor of  claim 16 , wherein the enzyme is caspase. 
     
     
         19 . The biosensor of  claim 16 , wherein the fluorescent protein comprises green fluorescent protein, red fluorescent protein, or yellow fluorescent protein. 
     
     
         20 . A biosensor comprising a nucleic acid comprising a stop codon cassette flanked by site specific recombinase recombination sequences, wherein the stop codon cassette and flanking sequences separate a constitutive promoter and a fluorescent protein open reading frame. 
     
     
         21 . The biosensor of  claim 20 , wherein the recombinase is a recombinase expressed only during apoptosis.