Genetically encoded cell death indicators and methods of use
AssigneeThe J. David Gladstone Institutes, a Testamentary Trust Established Under the Will of J. David Glads
InventorsJeremy Linsley, Kevan Shah, Steven M. Finkbeiner
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Abstract
Genetically encoded death indicator (GEDI) polypeptides and nucleic acid molecules encoding such polypeptides are provided. In addition, methods of using such nucleic acids and polypeptides to monitor cell death events in vitro and in vivo, particularly in neuronal cell death, are also provided.
Claims19
A method for monitoring cell death in a cell comprising an isolated nucleic acida nucleotide sequence encoding:
- a. a promoter;
- b. a first detectable marker;
- c. a modified calcium binding motif modified to reduce calcium binding affinity;
- and
- d. a second detectable marker;
- wherein the method comprises:
- a. measuring a signal from the first detectable marker to generate a first signal intensity;
- b. measuring a signal from the second detectable marker to generate a second signal intensity;
- c. comparing the first signal intensity to the second signal intensity to generate a signal intensity ratio;
- and
- d. determining whether the cell is dead or alive based on the signal intensity ratio.
A method of screening for an agent that is capable of causing an intracellular calcium concentration fluctuation in a cell comprising:
- a. contacting a cell with the agent;
- b. measuring a signal from the first detectable marker to generate a first signal intensity;
- c. measuring a signal from the second detectable marker to generate a second signal intensity;
- d. comparing the first signal intensity to the second signal intensity to generate a signal intensity ratio;
- and
- e. determining whether the agent is capable causing an intracellular calcium concentration fluctuation based the signal intensity ratio;
- wherein the cell comprises a nucleotide sequence encoding:
- a. a promoter;
- b. a first detectable marker;
- c. a modified calcium binding motif of SEQ ID NO. 1 or variants thereof;
- and
- d. a second detectable marker.
Dependencies are inferred from the claim text.