Constructs for gene expression analysis

Abstract

The present invention relates generally to constructs and their use in gene expression or gene regulation assays. More particularly, the present invention provides expression vectors and/or reporter vectors providing kinetics of protein expression with improved temporal correlation to promoter activity. Even more particularly, the invention provides expression vectors comprising a transcribable polynucleotide which comprises a sequence of nucleotides encoding a RNA element that modulates the stability of a transcript corresponding to the transcribable polynucleotide. The present invention provides, inter alia, novel vectors, useful for identifying and analysing cis- and trans-acting regulatory sequences/factors as well as vectors and genetically modified cell lines or organisms that are particularly useful for drug screening and drug discovery.

1 - 22 . (canceled) 
     
     
         23 . A recombinant DNA construct for assaying the activity of a gene expression-modulating element or for identifying elements of this type or agents that modulate their activity, the construct comprising in operable linkage a polynucleotide that encodes a reporter polypeptide and one or more heterologous nucleic acids sequences that encode destabilizing elements selected from the group consisting of protein destabilizing element and mRNA destabilizing element, such that the reporter polypeptide has an effective half-life of less than about 5 hours in a eukaryotic cell, wherein the construct either comprises or lacks but comprises a site for introducing, the gene expression-modulating element in operable connection with the polynucleotide, wherein the effective half-life is the rate of decay of the reporter polypeptide after a block in transcription as a result of the combined effects of protein stability and mRNA stability. 
     
     
         24 . The construct of  claim 23 , wherein the gene expression modulating element is a transcription modulating element or a post-transcriptional control element. 
     
     
         25 . The construct of  claim 23 , wherein following the block in transcription, the level of the polypeptide is reduced by at least 50% within about 5 hours, about 3 hours, about 2 hours, or about 1 hour. 
     
     
         26 . The construct of  claim 23 , wherein a level of expression of the polypeptide provides an indicator of the activity of the gene expression-modulating element. 
     
     
         27 . The construct of  claim 23 , wherein the one or more heterologous nucleic acid sequences encode a mRNA destabilizing element in operable linkage with the polynucleotide, where the mRNA destabilizing element reduces the stability of a transcript encoded by the polynucleotide, wherein the polynucleotide and the nucleic acid sequences are heterologous to each other. 
     
     
         28 . The construct according to  claim 23 , wherein the one or more heterologous nucleic acid sequences encode a protein destabilizing element and are in operable linkage with the polynucleotide, wherein the protein destabilizing element reduces the stability of the reporter polypeptide, and wherein the polynucleotide and the nucleic acid sequences are heterologous to each other. 
     
     
         29 . The construct according to  claim 23 , wherein the protein destabilizing element is selected from a PEST sequence, an N-terminal destabilizing amino acid or an ubiquitin or a biologically active fragment thereof, or variant or derivative of these. 
     
     
         30 . The construct according to  claim 23 , wherein the reporter protein is selected from an enzymatic protein, a protein associated with the emission of light, a fluorescent protein or a luminescent protein. 
     
     
         31 . The construct according to  claim 30 , wherein the reporter protein is selected from Luciferase, Green Fluorescent Protein, Red Fluorescent Protein, SEAP, CAT, or biologically active fragments thereof, or variants or derivatives of these. 
     
     
         32 . The construct according to  claim 23 , wherein the gene expression-modulating element is a transcriptional control element. 
     
     
         33 . The construct according to  claim 32 , wherein the transcriptional control element is a promoter. 
     
     
         34 . The construct according to  claim 33 , wherein the promoter is a non-constitutive promoter. 
     
     
         35 . The construct according to  claim 34 , wherein the non-constitutive promoter is regulated by a cellular signal transduction pathway. 
     
     
         36 . The construct according to  claim 23 , wherein the gene expression-modulating element is a cis-acting regulatory element. 
     
     
         37 . A cell comprising a construct according to  claim 23 . 
     
     
         38 . A genetically modified non-human organism comprising one or more constructs according to  claim 23 . 
     
     
         39 . A method for identifying an agent that modulates the activity of a gene expression-modulating element, the method comprising:
 expressing under the control of the gene expression-modulating element a recombinant DNA construct comprising in operable linkage a polynucleotide that encodes a transcript that encodes a reporter polypeptide and one or more heterologous nucleic acid sequences that encode destabilizing elements selected from the group consisting of protein destabilizing element and mRNA destabilizing element, such that the reporter polypeptide has an effective half-life of less than about 5 hours in a eukaryotic cell in the presence and absence of a test agent; and   measuring and comparing the level or functional activity of the reporter polypeptide in the presence and absence of the test agent, wherein a difference between the level or functional activity of the polypeptide in the presence and absence of the test agent indicates that the test agent modulates the activity of the gene expression-modulating element;   wherein the effective half-life is the rate of decay of the polypeptide after a block in transcription as a result of the combined effects of protein stability and mRNA stability.   
     
     
         40 . The method of  claim 39 , wherein the gene expression-modulating element is a transcription modulating element or a post-transcriptional control element. 
     
     
         41 . The method of  claim 39 , wherein following the block in transcription, the level of the polypeptide is reduced by at least 50% within about 5 hours, about 3 hours, about 2 hours, or about 1 hour.