Compositions and methods for detecting intracellular glucose and analogs thereof

Abstract

The subject invention pertains to nucleic acid constructs for post-transcriptional control of expression of a polynucleotide encoding a protein in a cell, wherein the constructs include a metabolite responsive instability element such as the glucose-regulated mRNA instability element. The subject invention further pertains to host cells and vectors comprising the nucleic acid constructs of the invention, as well as probes, methods, and kits for detecting metabolite responsive instability elements or mutations thereof. The present invention further concerns a reporter vector useful for detecting intracellular glucose and glucose-analogs, host cells genetically modified with the reporter vector, and methods for detecting intracellular glucose. The present invention utilizes an element that regulates messenger RNA (mRNA) stability in response to a metabolite such as glucose or a glucose analog. This glucose-regulated mRNA instability element has been mapped to the protein kinase C βII (PKCβII) mRNA that was found to decrease in the presence of elevated glucose levels. When cloned into a reporter vector, the region of PKCβII containing the mRNA instability element imparts glucose-sensitive instability to the mRNA that is transcribed, thereby down-regulating the expression of the reporter gene when glucose is elevated. The reporter vector of the present invention may be introduced into host cells, allowing detection of intracellular glucose and glucose analogs within intact, living cells in real-time and, optionally, in a high-throughput format.