Methods for identifying novel nucleic acid regulatory elements and compounds that affect the regulation
OwnerAnthony Giordano, Paul S. Eder +2inventors
InventorsAnthony Giordano, Paul S. Eder, Deborah Beach McGill, Gregg Johannes
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Abstract
Described herein are methods for identifying novel nucleic acid regulatory elements and compounds that modulate the regulation of such elements. Also described herein are nucleic acid sequence identified as novel nucleic acid regulatory elements and host cells containing such nucleic acid regulatory elements in a vector.
Claims17
A method of identifying one or more candidate compounds having an effect on the translation of a gene of interest, said method comprising:
- (a) identifying a 5′ or 3′ UTR sequence from a gene of interest that regulates translation;
- (b) selecting host cells which express the gene that is endogenously associated with the UTR;
- (c) stably transfecting one set of host cells with a test expression vector, said test vector comprising said 5′ or 3′ UTR sequence that regulates translation linked to, and positioned upstream or downstream, respectively, of a reporter gene construct (UTR/reporter), and stably transfecting another set of host cells with a control expression vector, said control vector comprising said reporter gene and lacking said UTR sequence (control/reporter);
- (d) identifying whether said UTR sequence has a translational function by assessing whether a first mRNA function of the UTR/reporter transcript is changed compared to the control/reporter transcript;
- (e) determining whether the UTR regulatory function identified in step (d) is physiologically relevant by assessing whether said changed first mRNA function of the UTR/reporter transcript corresponds to the first mRNA function of the gene endogenously associated with the UTR;
- (f) contacting said compound with an mRNA molecule comprising said UTR sequence;
- and
- (g) measuring whether said compound alters a UTR-mediated translational effect.
A method of identifying one or more candidate compounds having an effect on translation through the modulation of an intracellular pathway-mediated involved in UTR translational regulation, said method comprising:
- (a) identifying a 5′ or 3′ UTR sequence from a gene of interest that regulates translation;
- (b) selecting host cells which express the gene that is endogenously associated with the UTR;
- (c) stably transfecting one set of host cells with a test expression vector, said test vector comprising said 5′ or 3′ UTR sequence that regulates translation linked to, and positioned upstream or downstream, respectively, of a reporter gene construct (UTR/reporter), and stably transfecting another set of host cells with a control expression vector, said control vector comprising said reporter gene and lacking said UTR sequence (control/reporter);
- (d) identifying whether said UTR sequence has a translational function by assessing whether a first mRNA function of the UTR/reporter transcript is changed compared to the control/reporter transcript;
- (e) determining whether the UTR regulatory function identified in step (d) is physiologically relevant by assessing whether said changed first mRNA function of the UTR/reporter transcript corresponds to the first mRNA function of the gene endogenously associated with the UTR;
- (f) contacting said compound with an mRNA molecule comprising said UTR sequence;
- and
- (g) measuring whether said compound alters an intracellular pathway-mediated UTR translational effect.
Dependencies are inferred from the claim text.