| REPORTER GENE EXPRESSION IN SINGLE, LIVING CELLS BY PHOTON COUNTING IMAGING OF LUCIFERASE REPORTER GENE. |
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Reporter gene expression is based in the introduction into cells of plasmids containing a reporter gene under control of a promoter, promoter region or response element. Upon activation of promoter by specific transcription factors, the reporter gene is expressed and measurement of its activity allows to estimate transcription activity of the promoter, promoter region or transcription factor acting on a particular response element. The most popular reporter gene is luciferase, a bioluminescent enzyme that produces emission light (Bioluminescence) in the presence of ATP and luciferin. Luciferase reporter gene activity can be now monitored in single, living cells by the use of high-sensitive photon counting cameras enabling monitoring dynamics of gene expression or transcriptional activity in living cells. We have used the new method for monitoring changes in prolactin gene expression, GnRH gene expression or transcriptional activity of topoisomerase II alpha along the cell cycle. In this latter case, we have monitored concurrently both transcriptional activity and cell division by combining bioluminescence and bright field imaging. For this end, NIH 3T3 cells stably transfected with the luciferase reporter gene under control of the topoisomerase-IIa gene promoter (Falck et al. 1999) were synchronised in medium containing 0,5% serum for 3 days. Then, 10% serum and 1 mM luciferin was added and the Petri dish containing the cells was placed in an incubator (Zeiss CTI-controller 3700) controlling temperature, CO2 and humidity attached over the stage of an inverted microscope (Zeiss Axiovert 100 TV). Photonic emissions and bright field images were captured concurrently with a Hamamatsu VIM photon counting camera handled with an Argus-20 image processor at 15 min intervals for 72 hours. Transcription activity is expressed as total photonic emissions in each image minus background photonic emissions.
Photonic emissions are driven by the topoisomerase II alpha promoter. This gene is activated during S phase of the cell cyle and its transcriptional activity was measured every 15 min for 72 h. Bright field images of the same cells were captured concurrently to monitor simultaneously gene transcription and cell division in the same cells. Trace below shows light emission of the microscopic field during the measurement period. |
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Cell proliferation depends on mitochondrial Ca2+ uptake: inhibition by salicylate. |
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| Núñez L, Valero RA, Senovilla L, Sanz-Blasco S, García-Sancho J, Villalobos C (2006). J Physiol 571, 57-73. |
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Calcium dynamics and resting transcriptional activity regulates prolactin gene expression. |
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| Villalobos C, Núñez L, Faught WJ, Leaumont DC, Boockfor FR, Frawley LS (2002). Endocrinology 143, 3548-3554. |
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