Methods and compositions for identifying and validating modulators of cell fate
AssigneeChromocell Corporation
InventorsKambiz Shekdar, Dennis J. Sawchuk, Jessica C. Langer
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Abstract
The invention provides for compositions and methods for identifying and validating modulators of cell fate, such as such as maintenance, cell specification, cell determination, induction of stem cell fate, cell differentiation, cell dedifferentiation, and cell trans-differentiation. The invention relates to reporter nucleic acid constructs, host cells comprising such constructs, and methods using such cells and constructs. The invention relates to methods for making cells comprising one or more reporter nucleic acid constructs using fluorogenic oligonucleotides. The methods relate to high throughput screens.
Claims19
An in vitro method for making a recombinant cell comprising the steps of:
- (a) introducing into a cell a recombinant nucleic acid construct comprising:
- i) an open reading frame (ORF) encoding a reporter wherein the ORF is operably linked to a cell type related (CTR) promoter, and wherein the CTR promoter is not active above background levels in the cell;
- and
- ii) a nucleic acid sequence encoding a first target sequence RNA1 (TSR1), a nucleic acid sequence encoding a second target sequence RNA2 (TSR2), and a nucleic acid sequence encoding a third target sequence RNA3 (TSR3), wherein the TSR3 is cotranscribed with the reporter, and wherein the TSR1 and TSR2 flank the reporter ORF and the CTR promoter, and wherein the transcription of TSR1 and TSR2 are each driven by a constitutively active promoter that is different from the CTR promoter;
- (b) introducing into the cell fluorogenic oligonucleotides that are complementary to TSR1, TSR2, and TSR3;
- and
- (c) selecting cells that transcribe both TSR1 and TSR2, and do not transcribe TSR3 above background levels.
Dependencies are inferred from the claim text.