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Artificial transcription factors targeting any desired genes are very attractive from the standpoint of regulating biological functions for life science studies and clinical applications. In order to generate such transcription factors, specific DNA binding domains are required to address a single site for each gene promoter. C(2)H(2) type zinc finger motif is one of the best frameworks to create new artificial DNA binding proteins for the following features: the zinc finger motif can recognize three bases DNA, be tandemly repeated by covalent linkage, and work as a monomer. Taking advantage of these features, manifold zinc finger proteins targeting various DNA sequences have been created so far. For application to a target in sequences as complex as the human genome, the significantly strict specificity in DNA binding must be required. Conjugating multiple fingers (multi-zinc fingers) enables to recognize longer sequences which are sufficient for addressing a single site in the human genome, whereas it has become known that as the number of finger motifs increases, the equilibrium time with the target sequence is significantly longer by in vitro experiments. Our recent study showed that the multi-zinc finger type artificial transcription factor could activate the reporter gene promptly. There is much interest in creating gene regulators, and the artificial transcription factors based on multi-zinc finger motifs could be a superior scaffold.

The studies on the mechanism of HCV replication proliferated after the development of cell culture based-subgenomic HCV replicon system and genome-length HCV RNA replication system. Furthermore, these RNA replication systems have been improved to be suitable systems for the screening of anti-HCV reagents by the introduction of reporter genes such as luciferase. Genetic analysis of HCV RNAs obtained in long-term cell culture of HCV replicon or genome-length HCV RNA-harboring cells revealed that genetic mutations in HCV RNAs accumulated in a time-dependent manner. The genetic diversity of HCVs was also enlarged in a time-dependent manner. The appearance of adaptive mutation in HCV replicon or genome-length HCV RNA is one of characteristic features of HCV RNA replication system. Although human hepatoma-derived HuH-7 cell line was mainly used for HCV RNA replication systems, a specific combination of adaptive mutations led to develop the HCV RNA replication systems using a new human hepatoma cell line other than HuH-7.

We examined the systematic assay of the reporter gene for the assessment of heavy metals and organic chemical pollutants using the reporter plasmids carrying stress-responsive elements fused to the green fluorescence protein (GFP) gene as follows: metallothionein (MTIIA), heme oxigenase-1 (HO-1), quinone reductase (ARE), and c-fos genes. The treatment of COS7 cells in which the c-fos gene promoter-, ARE-, or HO-1 enhancer-fused GFP with a low concentration of NaAsO(2) was introduced led to the detection of the fluorescent cells, and an agrichemical paraquat enhanced the fluorescence of ARE or HO-1 enhancer-transfected cells. The cells in which the plasmid carrying the MT-IIA gene promoter (the -765 bp upstream from the transcription initiation site) was introduced highly expressed GFP on treatment with CdCl(2), ZnSO(4), or CuCl(2). The plasmid carrying seven metal-responsive elements of the MT-IIA gene increased the response of the fluorescence intensity to these heavy metals. These results indicated that the use of the gene promoters and enhancers of the stress-responsive genes fused to GFP contributes to the visualization of pollutant-responsive mammalian cells and can be applied to biomonitoring of environmental pollution.

Radionuclide gene expression imaging can visualize and measure transcription activity of target genes noninvasively by co-expressing a reporter gene, of which product can be traced with complementary radioactive reporter probes. While in the cardiac field it was at first aiming at monitoring angiogenic gene therapy, its application is directed to cell tracking in stem/progenitor cell therapy, due to disappointing results in the clinical trials and potential risks of transfer vectors. In this article, we give a general outline of cardiac radionuclide gene expression imaging using reporter genes, including its principle, various combinations of reporter gene/reporter probe, the current status of cardiac gene therapy, and its application for cell tracking.

The reporter gene expression method CALUX cell bioassay has proven to be a very valuable screening technique for assessing toxic equivalents of dioxin and dioxin-like compounds, because it detects all AhR ligands in a variety of sample matrices. Used with the two specially elaborated clean-up procedures, CALUX cell bioassay was established for various environmental and biological samples. Consequently, the CALUX-TEQ values are correlated with high correlation coefficiencies to WHO-TEQ values derived from HRGC/HRMS. CALUX assay has been applied to the screening and quarantine of food items with possible dioxin contaminations in the EU. In addition, the method has been applied to biological samples such as serum, breast milk and fat tissues. Several reported studies demonstrated that CALUX cell assay is a useful tool and applicable in human epidemiological studies. We need to establish multi-national comparative studies in the field of dioxin contamination.

The genetic reporter system has been introduced as a tool for sensitive and convenient monitoring of gene expression. In the present study, the promoter activity of growth hormone (GH)-coding gene was sequentially monitored in GH3 cells (rat pituitary adenoma cell-line) by means of bioluminescence which was evoked by a secreted reporter, Vargula hilgendorfii (VH) luciferase. Three kinds of expression plasmids were constructed, which consisted of a rat GH promoter fragment and VH luciferase cDNA, and transfected into GH3 cells. The sensitivity of the reporter was revealed to be extremely high from the dose-response curve of VH luciferase. With the use of the stable transformant, the reporter activity as well as that of GH was sequentially measured in plate culture. The intracellular dynamics of reporter signals was analyzed through the response to inhibitors of transcription, protein synthesis and vesicle transport, which suggested that the overall time from transcription to secretion was essentially identical to those of the VH luciferase and GH although the both proteins took different time course at different intracellular stages. When the GH promoter was activated by triiodothyronine (T3), the reporter activity was enhanced, and well correlating with the amount of GH mRNA, which indicated a reliability of the reporter system. Furthermore, in the present reporter system, a synergistic effect of T3 and dexamethasone was observed not only on the GH transcription but also on the stability of mRNA. It is concluded that the bioluminescence reporter system using the VH luciferase is an excellent tool for sequential monitoring of the GH transcriptional activity.

Transcriptional activation of the expression of the gene for interleukin (IL)-8) in airway epithelial cells was analyzed by stable transfection of human bronchial epithelial cells (16 HBE) with a reporter plasmid pIL-8/Luc, which the expression of luciferase reporter gene is driven by human IL-8 promoter. As compared with the resting condition, the luciferase activity was 3.9 times higher after stimulation with phorbol-myristate-acetate (20 ng/ml), 1.6 times higher after stimulation with tumor necrosis factor-alpha (100 U/ml), and 2.7 times higher after stimulation with interleukin-1 beta (100 U/ml). Dexamethasone inhibited the effects of these stimulants by 10 to 50 percent. These results closely correspond to those of IL-8 mRNA analyses with Northern blotting and IL-8 protein analyses done by enzyme-linked immunosorbent assay. The transgenic cell line IL-8 Luc/16HBE can also be used in screening for drug effects, and may be useful for quantifying IL-8 inducibility in clinical samples obtained from the lungs.