Abstract:Firefly luciferase is used widely as a reporter enzyme for studies of gene regulation and expression. The recent development of new technologies that combine luciferase reporter technology and digital imaging microscopy has enabled multiple measurements of gene expression in the same living cell. Although this approach has already provided new insights about expression dynamics, its future utility is limited by the three- to four-hour half-life of firefly luciferase in mammalian cells. Because of this, rapid i… Show more
“…A slight difference in decay of luciferase activity and hTERT mRNA levels might be due to different stability of luciferase mRNA (half-life ~4-10h in human cells) and hTERT mRNA (half-life ~1h in human cells). [26][27][28] As the 0.3 kbp fragment was repressed during HepaRG cell differentiation we aimed at identifying the regulatory sequences which might be responsible for hTERT promoter activity in hepatocyte proliferation. For this purpose we used hTERT core promoter constructs with mutations ( Fig.…”
Section: Defining Regulatory Elements Controlling Htert Expression Inmentioning
“…A slight difference in decay of luciferase activity and hTERT mRNA levels might be due to different stability of luciferase mRNA (half-life ~4-10h in human cells) and hTERT mRNA (half-life ~1h in human cells). [26][27][28] As the 0.3 kbp fragment was repressed during HepaRG cell differentiation we aimed at identifying the regulatory sequences which might be responsible for hTERT promoter activity in hepatocyte proliferation. For this purpose we used hTERT core promoter constructs with mutations ( Fig.…”
Section: Defining Regulatory Elements Controlling Htert Expression Inmentioning
“…As shown in Figure 7, the stable transfectant exhibited comparable luciferase activity in all phases. Considering the short half-life of luciferase protein in cells (approximately 2~3 h) [27][28][29], the intrinsic activity of transcription factors does not appear to be affected by the cell cycle.…”
Section: Effect Of Cell Cycle On the Intrinsic Activity Of Transcriptmentioning
“…Details of the methodology have been previously described. 64 To determine vectors titers (TU per milliliter or TU ml -1 ), 2-2 cells were infected and green fluorescent cells were counted under a Nikon Eclipse TE300 inverted fluorescence microscope (Nikon, Tokyo, Japan) after 24 h.…”
Hepatocellular carcinoma (HCC) is usually refractory to the available treatments. For cancer gene therapy purposes, real-time imaging of therapeutic gene expression is of great importance because there are multiple factors that modulate the therapeutic gene expression in a complex tumor microenvironment. As a consequence, multiple doses of therapeutic viral vectors may be required for improved efficacy. In the present study, the luciferase reporter gene and the yeast cytosine deaminase (yCD) genes were bicistronically expressed using the foot-and-mouth disease virus 2A peptide under the regulation of the cytomegalovirus (CMV) promoter. The effectiveness of the yCD/5-FC (5-fluorocytosine) killing efficacy mediated by the herpes simplex virus type 1 (HSV-1) amplicon viral vector was shown using HCC and non-HCC cell lines in vitro. In addition, in vivo experiment also showed tumor regression of a primary HCC 26-1004 tumor xenograft in tumor expressing high levels of the yCD gene (as determined by noninvasive imaging) after intratumoral injection of 1.5Â10 6 TU HGCX-L2C HSV-1 amplicon viral vector and 5-FC administration. The HSV-1 amplicon viral vector coupled with the yCD/5-FC prodrug activated suicide gene could potentially be of use in clinical gene therapy for HCC.
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