Comparative activity of several promoters in driving NIS expression in melanoma cells

Targeted drug delivery systems are special importance for developing gene therapeutic drugs that recognize and eliminate tumor cells. It is desirable that therapeutic genes be expressed predominantly in tumor cells after their targeted delivery into the tumor. Hence, the distribution of the expression product through various tissues should be studied when testing a therapeutic gene in vivo. The sodium iodide symporter (NIS) is attractive as a reporter because its tissue level is easy to quantify by noninvasive imaging methods. Therapeutic gene expression in tumor cells is achieved using various promoters, including strong nonspecific promoters; moderately active tissue-specific promoters; and tumor-specific promoters, which function in a broad range of tumor cells, but have low activity. The relationship between the promoter strength and reporter NIS activity is still unclear. The reporter gene was used to test three promoters types for activity in melanoma cells. The functional activity of NIS expressed from a cloned gene was compared for the three promoters types. Although the promoters greatly varied in strength, only minor changes were observed for NIS functional activity. A relatively weak melanoma-specific promoter ensured a high NIS activity in melanoma cells. Weaker tumorspecific promoters determined a high NIS activity only in some cells of the melanoma origin.     

The sea pansy Renilla reniformis luciferase serves as a sensitive bioluminescent reporter for differential gene expression in Candida albicans.

The infectious yeast Candida albicans progresses through two developmental programs which involve differential gene expression, the bud-hypha transition and high-frequency phenotypic switching. To understand how differentially expressed genes are regulated in this organism, the promoters of phase-specific genes must be functionally characterized, and a bioluminescent reporter system would facilitate such characterization. However, C. albicans has adopted a nontraditional codon strategy that involves a tRNA with a CAG anticodon to decode the codon CUG as serine rather than leucine. Since the luciferase gene of the sea pansy Renilla reinformis contains no CUGs, we have used it to develop a highly sensitive bioluminescent reporter system for C. albicans. When fused to the galactose-inducible promoter of GAL1, luciferase activity is inducible; when fused to the constitutive EF1 alpha 2 promoter, luciferase activity is constitutive; and when fused to the promoter of the white-phase-specific gene WH11 or the opaque-phase-specific gene OP4, luciferase activity is phase specific. The Renilla luciferase system can, therefore, be used as a bioluminescent reporter to analyze the strength and developmental regulation of C. albicans promoters.     

Cell cycle dependent TN-C promoter activity determined by live cell imaging

The extracellular matrix protein tenascin-C plays a critical role in development, wound healing, and cancer progression, but how it is controlled and how it exerts its physiological responses remain unclear. By quantifying the behavior of live cells with phase contrast and fluorescence microscopy, the dynamic regulation of TN-C promoter activity is examined. We employ an NIH 3T3 cell line stably transfected with the TN-C promoter ligated to the gene sequence for destabilized green fluorescent protein (GFP). Fully automated image analysis routines, validated by comparison with data derived from manual segmentation and tracking of single cells, are used to quantify changes in the cellular GFP in hundreds of individual cells throughout their cell cycle during live cell imaging experiments lasting 62 h. We find that individual cells vary substantially in their expression patterns over the cell cycle, but that on average TN-C promoter activity increases during the last 40% of the cell cycle. We also find that the increase in promoter activity is proportional to the activity earlier in the cell cycle. This work illustrates the application of live cell microscopy and automated image analysis of a promoter-driven GFP reporter cell line to identify subtle gene regulatory mechanisms that are difficult to uncover using population averaged measurements.