Patent ID: 9663833
Date: 2017-05-30
CPC Classifications: C12Q

Claim:
1. A process of assessing gene expression in a cell or in a cell line wherein the assessed gene is an endogenous gene, comprising: (a) providing a cell or cell line wherein a double-stranded polynucleotide construct has been introduced, which double-stranded polynucleotide construct is suitable for performing gene expression assay when integrated into a cell which naturally harbours and expresses gene(s) of interest for an activity, and which comprises on its positive strand considered from its 5′ end to its 3′ end, (i) a promoter of a gene of interest or several promoters of various genes of interest selected among genes which are endogenous to the cell and subject to gene transcription profiling, wherein said promoter is recognized by the internal transcription machinery of the cell, and, (ii) one or several beacon barcode(s) wherein each barcode contains at least one barcode unit which is a DNA construct comprising tandem repeats of at least one beacon recognition binding site each binding site being composed of a nucleotide sequence, and wherein each of said barcode(s) is(are) under the control of one of said at least one promoter(s) for transcription, (b) eliciting, silencing or modulating transcription of the polynucleotide construct (c) contacting the cell or cell line of step (a) with detection probe(s) capable of hybridizing with the beacon recognition binding site(s) of the barcode(s) and which is (are) one or several molecular beacon(s), said molecular beacon(s) having a stem-and-loop polynucleotide structure and being suitable for visualisation or measurement when hybridized to their target sequence, wherein the visualisation of the hybridization of the detection probe(s) with their target is obtained as a result of fluorescence which is switched on when the detection probe binds to its target sequence, (d) detecting hybridization between the detection probes and the transcript of the beacon recognition binding sites of the barcode as a reporter of transcription activity of the promoter of the polynucleotide construct, (e) measuring gene expression in the cell or in the cell line of step (a) on the transcriptional level, by quantifying in vivo hybridization events of the molecular beacon(s) with the transcript of the polynucleotide construct resulting in an increase in fluorescence obtained in step (c) over the signal of said molecular beacon(s) not hybridized to their target sequence.