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Hypoxia activates constitutive luciferase reporter constructs.

ABSTRACT: Hypoxia has been identified as a contributing factor in the pathophysiology of several diseases and oxygen regulation is important during stem cell development, particularly in early embryogenesis. One aspect that has emerged is the role of hypoxia-inducible factors, or HIFs in regulating the effect of hypoxia. Studies in our laboratory sought to examine the hypoxic regulation of HIF activity in placental trophoblast cells, through the use of dual-reporter luciferase assays. Our study demonstrates that hypoxic conditions cause a significant increase in the level of constitutive luciferase reporter activity. We also show that this induction is not a cell type or species-specific phenomenon and provides an alternative method for normalizing transfection efficiency in luciferase assays under hypoxic conditions. Our results suggest that in studies dealing with hypoxic conditions, caution should be used when interpreting measurements of transcriptional activity by traditional dual-reporter assays.

SUBMITTER: Doran DM

PROVIDER: S-EPMC3020074 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Hypoxia activates constitutive luciferase reporter constructs.

Doran Diane M DM Kulkarni-Datar Kashmira K Cool David R DR Brown Thomas L TL

Biochimie 20101026 2

Hypoxia has been identified as a contributing factor in the pathophysiology of several diseases and oxygen regulation is important during stem cell development, particularly in early embryogenesis. One aspect that has emerged is the role of hypoxia-inducible factors, or HIFs in regulating the effect of hypoxia. Studies in our laboratory sought to examine the hypoxic regulation of HIF activity in placental trophoblast cells, through the use of dual-reporter luciferase assays. Our study demonstrat ...[more]

PMID: 20971156

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Project description:BackgroundDown-regulation or silencing of transgene expression can be a major hurdle to both molecular studies and biotechnology applications in many plant species. Sugarcane is particularly effective at silencing introduced transgenes, including reporter genes such as the firefly luciferase gene.Synthesizing transgene coding sequences optimized for usage in the host plant is one method of enhancing transgene expression and stability. Using specified design rules we have synthesised new coding sequences for both the firefly luciferase and Renilla luciferase reporter genes. We have tested these optimized versions for enhanced levels of luciferase activity and for increased steady state luciferase mRNA levels in sugarcane.ResultsThe synthetic firefly luciferase (luc*) and Renilla luciferase (Renluc*) coding sequences have elevated G + C contents in line with sugarcane codon usage, but maintain 75% identity to the native firefly or Renilla luciferase nucleotide sequences and 100% identity to the protein coding sequences.Under the control of the maize pUbi promoter, the synthetic luc* and Renluc* genes yielded 60x and 15x higher luciferase activity respectively, over the native firefly and Renilla luciferase genes in transient assays on sugarcane suspension cell cultures.Using a novel transient assay in sugarcane suspension cells combining co-bombardment and qRT-PCR, we showed that synthetic luc* and Renluc* genes generate increased transcript levels compared to the native firefly and Renilla luciferase genes.In stable transgenic lines, the luc* transgene generated significantly higher levels of expression than the native firefly luciferase transgene. The fold difference in expression was highest in the youngest tissues.ConclusionsWe developed synthetic versions of both the firefly and Renilla luciferase reporter genes that resist transgene silencing in sugarcane. These transgenes will be particularly useful for evaluating the expression patterns conferred by existing and newly isolated promoters in sugarcane tissues. The strategies used to design the synthetic luciferase transgenes could be applied to other transgenes that are aggressively silenced in sugarcane.

Dataset Information

Hypoxia activates constitutive luciferase reporter constructs.

Publications

Hypoxia activates constitutive luciferase reporter constructs.

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