KR20030073087A - Positive Cell Based Assay System Using Repressor like HDAC - Google Patents

Abstract

PURPOSE: Provided is a cell based assay system which inhibits the expression of a reporter by the activation of a repressor, and induces the expression of the reporter upon receipt of proper signals, thereby evaluating the given signals. CONSTITUTION: A specific inhibition regulating gene and a constant expression gene are constructed before the target gene such that the target gene is constantly expressed under the normal circumstances, but specifically repressed by a specific repressor. The activity of histone deacetylase(HDAC) is operated as the specific repressor. The activity of the HDAC within the cell is traced using a vector with the HDAC fused at the DNA binding domain.

Description

Positive Cell Based Assay System Using Repressor like HDAC}

The present invention is to evaluate the various activities in the cell by inducing the expression of the reporter gene, conventionally in the normal state by attaching a reporter under the promoter that is transcribed by the gene to analyze the activity or a protein that is an expression product thereof. The reporter is expressed, and the expression of the reporter gene is suppressed by inhibiting the activity of the target protein. That is, a negative analysis method for confirming that the activity of the target protein is inhibited by suppressing the expression of the reporter gene has been widely used. The conventional voice analysis method has always been concerned that false positives are more than an error or an error in experiments, and it has been recognized that problems leading to unnecessary resource consumption in the application of a large-scale drug discovery method have been recognized. .

The present invention aims to develop a system for expressing a reporter by a specific signal in a cell which can reduce the time and cost required for drug development and increase the convenience and accuracy of the analysis method by overcoming these problems.

In the present invention, first, to determine whether it is possible to specifically inhibit the expression of the reporter when using a transcription inhibitory factor. In other words, HAC (HDAC), one of transcription inhibitory factors, was used to construct a gene combination that can specifically inhibit the expression of reporter genes in cells.

Second, we tried to determine whether inhibition of reporter expression by the transcription inhibitor HDAC can be restored by inducing the expression of the reporter gene by inhibiting the activity of the transcription inhibitor HDAC. In other words, it was confirmed that the inhibition of HDAC activity can be confirmed by the expression of the reporter gene.

Figure 1a is a schematic diagram showing the gene carrier gene configuration to fuse the DNA binding site and transcription inhibitory factor

Figure 1B is a schematic diagram showing the gene composition of a reporter gene carrier characterized by transcription inhibition in the presence of specific transcription inhibitory factors.

Figure 2 shows the results of transcription inhibitory activity of luciferase according to TSA treatment after introducing the gene carriers pBGal4Hdac and pR-Luc into HeLa cells.

Figure 3 is a schematic diagram showing the gene composition of the Gal4-Rb fusion gene carrier designed to track the activity of HDAC in the cell

The present invention is a gene carrier designed to express a fused repressor capable of binding to a specific DNA sequence, and a reporter gene carrier designed to be specifically transcriptionally inhibited by a specific transcription repressor but always expressed under normal circumstances. It is configured to control the expression of the reporter gene by introducing into a cell.

In order to realize the present invention, a DNA binding protein capable of binding to a specific site of DNA was required, and for this purpose, a DNA binding site of the GAL4 protein of yeast was used. In addition, as a regulatory gene element to which the GAL4 binding site binds, a Gal1 promoter, that is, a GAL4 binding site was used. Luciferase was used as a reporter.

First, 5'-gal4 db / Hind III (GGGG AAG CTT GGG ATG AAG CTA CTG TCT TCT ATC), 3'-gal4 db / BamH I (GGGG GGA TCC CGA TAC) to clone the GAL4 protein binding domain (GAL4 binding domain) AGT CAA CTG TCT TTG) Two primers were synthesized and cloned by using Hind III, BamH I restriction enzyme under CMV promoter of pB (pvuII deleted pcDNBA3) carrier by PCR using these two primers. It was named pB-Gal4. PCR was also performed using two primers: 5'-HDAC / BamH I (GGGG GGA TCC GGG ATG GCG CAG ACG CAG GGC ACC) and 3'-HDAC / Not I (GGGG GCG GCC GCT CAG GCC AAC TTG ACC TCC TC). After performing, as shown in FIG. 1A, the HDAC1 gene was cloned to be fused with a GAL4 binding site and named pBGal4Hdac.

Reporter gene carriers may be used to convert Gal1 regulatory genes to 5'-Pgal1 / Bgl II (GGGG AGA TCT CCC CAT TAT CTT AGC CTA AAA AAA CCT), 5'-Pgal1 / MluI (GGGG ACG CGT CCT CCT TGA CGT TAA AGT ATA GAG G) Using the two primers of the clone was cloned using two restriction enzymes BglII, MluI of pcDNA3 and named pR. As shown in FIG. 1B, the reporter gene luciferase was cloned into the gene carrier using Hind III and NotI restriction enzymes and named pR-Luc.

As shown in Fig. 2, when the expression of the reporter gene, luciferase, was measured in the expression of the reporter gene Luciferase in only the gene carrier and the HeLa and HEK293 cell lines and the gene carrier and the pBGal4-Hdac carrier simultaneously. When the activity of the luciferase was added at the same time, it was confirmed that the expression was suppressed to about 20% compared to the control group without the fused transcription factor. In addition, luciferase, whose expression was specifically inhibited by the fused transcription factor Gal4-Hdac, inhibited the inhibitory activity of GAL4-Hdac by treating TSA, an inhibitor of HDAC, at a concentration of 100 ng / ml. The expression of luciferase was seen to be induced to 75% level.

As shown in FIG. 3, cloning Rb instead of HDAC in the fused GAL4-Hdac of FIG. 1A can track the activity of binding of HDAC and Rb in cells by measuring the amount of luciferase, a reporter. Can be used as an analytical system.

According to the present invention, by constructing a gene carrier to be constituve expression under normal circumstances and specifically to be transcriptionally inhibited by a transcription inhibitory factor, a cell-level activity evaluation system for positively analyzing the activity of HDAC in a cell was made. This allows for the screening of HDAC inhibitors and the suppression of reporter expression by placing HDACs in the GAL1 regulatory genes according to the binding properties of Rb and HDACs, such as using Gal4-Rb fusion proteins. And HDAC binding activity can be traced by measuring the activity of the reporter.

Claims (4)

A method of constructing a specific inhibitory regulatory gene and a homeostatic expression gene before the target gene so that the gene of interest is always expressed under normal circumstances and specifically transcriptionally inhibited by a specific transcription inhibitory factor. The method of specific transcription inhibition according to claim 1, which uses the activity of HDAC as a specific transcription inhibitory factor. Cell level activity evaluation method that can track the activity of HDAC in the cell by using the gene carrier comprising HDAC fused to the DNA carrier and the gene carrier constituted by the method of claim 1 Cell-level activity evaluation method that can track cell-specific HDAC activity in cells using gene carrier comprising Rb fused to DNA carrier of claim 2