Fluorescence amplification method for forward genetic discovery of factors in human mRNA degradation
AssigneeYale University
InventorsAndrei Alexandrov, Joan Steitz
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Abstract
The present invention provides systems and methods for performing forward genetic screens in cells and methods of treating disease through inhibiting targets identified in a forward genetic screen as involved in a pathway associated with the disease. The invention involves the use of multiple tandem fluorescent reporter molecules separated by cleavage sites under the control of a single promoter to enhance the fluorescent readout from a cell screen.
Claims9
A composition comprising a tandem reporter construct, wherein the tandem reporter construct comprises a single promoter operably linked to at least five tandem reporter genes for providing a detectable signal, and a nucleotide sequence encoding a protease, wherein each of the at least five tandem reporter genes are separated by protease cleavage sequences, and further wherein the nucleotide sequence encoding the protease comprises an internal protease cleavage site, and further wherein the tandem reporter construct comprises the nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
An isolated cell comprising at least two expression vectors, wherein each of the at least two expression vectors comprises a tandem reporter construct, wherein the tandem reporter construct comprises a single promoter operably linked to two or more tandem reporter genes for providing a detectable signal, and a nucleotide sequence encoding a protease, wherein each of the two or more tandem reporter genes are separated by protease cleavage sequences, and further wherein the nucleotide sequence encoding the protease comprises an internal protease cleavage site, wherein the cell is selected from the group consisting of:
- a) a cell transfected with a first expression vector and a second expression vector, wherein the first expression vector comprises the nucleotide sequence as set forth in SEQ ID NO:3, and wherein the second expression vector comprises the nucleotide sequence as set forth in SEQ ID NO:4, and
- b) a cell transfected with a first expression vector and a second expression vector, wherein the first expression vector comprises the nucleotide sequence as set forth in SEQ ID NO:2, and wherein the second expression vector comprises the nucleotide sequence as set forth in SEQ ID NO:5.
A composition comprising at least one tandem reporter construct, wherein the at least one tandem reporter construct comprises a single promoter operably linked to two or more tandem reporter genes for providing a detectable signal, and a nucleotide sequence encoding a protease, wherein each of the two or more tandem reporter genes are separated by protease cleavage sequences, and further wherein the nucleotide sequence encoding the protease comprises an internal protease cleavage site, wherein the at least one tandem reporter construct comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
Dependencies are inferred from the claim text.