The Luc2 gene enhances reliability of bicistronic assays

Mašek, Tomáš; Vopálenský, Václav; Pospíšek, Martin

doi:10.2478/s11535-013-0151-z

Cited by 6 publications

(3 citation statements)

References 20 publications

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“…Different substrates ensure minimal cross talk and spectral un-mixing techniques can be used for identification of each system on images. Has numerous variants and is the most cited in studies with its most useful applicability being in vivo analysis due to its red-shifted emission spectra; quantum yield: 41% [19], [24], [52], [150], [151] Synthetic/Engineered systems • Split luciferases: the luciferase enzyme is split apart into its C-terminus and N-terminus fragments with a protein or peptide inserted in the middle. Activation of the enzyme and the associated light output is only accomplished when the fragment in the middle interacts with the protein of interest.…”

Section: )mentioning

confidence: 99%

Bioluminescence Imaging Applications in Cancer: A Comprehensive Review

AlSawaftah

Farooq

Dhou

et al. 2021

IEEE Rev. Biomed. Eng.

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Bioluminescence imaging (BLI), an optical preclinical imaging modality, is an invaluable imaging modality due to its low-cost, high throughput, fast acquisition times, and functional imaging capabilities. BLI is being extensively used in the field of cancer imaging, especially with the recent developments in geneticengineering, stem cell, and gene therapy treatments. The purpose of this paper is to provide a comprehensive review of the principles, developments, and current status of BLI in cancer research. This paper covers the fundamental BLI concepts including BLI reporters and enzyme-substrate systems, data acquisition, and image characteristics. It reviews the studies discussing the use of BLI in cancer research such as imaging tumor-characteristic phenomena including tumorigenesis, metastasis, cancer metabolism, apoptosis, hypoxia, and angiogenesis, and response to cancer therapy treatments including chemotherapy, radiotherapy, immunotherapy, gene therapy, and stem cell therapy. The key advantages and disadvantages of BLI compared to other common imaging modalities are also discussed.

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Section: )mentioning

confidence: 99%

Bioluminescence Imaging Applications in Cancer: A Comprehensive Review

AlSawaftah

Farooq

Dhou

et al. 2021

IEEE Rev. Biomed. Eng.

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“…This short half-life yielded poor signal intensities of luciferase, which has made it very difficult to study particularly deep brain regions. The further modified reporter described herein eliminates this disadvantage by replacing the Luc gene with Luc2, which is codon-optimized for expression in mammalian cells (Mašek et al, 2013). As expected, Luc2 yielded brighter bioluminescence signals induced by acute stress at 4 h post-stress.…”

Section: Discussionmentioning

confidence: 99%

Improved dynamic monitoring of transcriptional activity during longitudinal analysis in the mouse brain

Heo

Jung

et al. 2018

Biology Open

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Bioluminescence imaging has proven to be a highly sensitive technique for assessing in vitro transcriptional activity toward understanding gene regulation patterns; however, application of this technique is limited for brain research. In particular, the poor spatiotemporal resolution is a major hurdle for monitoring the dynamic changes of transcriptional activity in specific regions of the brain during longitudinal analysis of living animals. To overcome this limitation, in this study, we modified a lentivirus-based luciferase glucocorticoid receptor (GR) reporter by inserting destabilizing sequence genes, and then the reporter was stereotaxically injected in the mouse infralimbic prefrontal cortex (IL-PFC). Using this strategy, we could successfully pin-point and monitor the dynamic changes in GR activity in IL-PFC during normal stress adaptation. The modified reporter showed a 1.5-fold increase in temporal resolution for monitoring GR activity compared to the control, with respect to the intra-individual coefficients of variation. This novel in vivo method has broad applications, as it is readily adaptable to different types of transcription factor arrays as well spanning wide target regions of the brain to other organs and tissues.

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“…Initial screening experiments for the comparison of different vehicles in Jurkat cells used either the luciferase (luc2 gene) (Mašek et al, 2013) expression plasmid pGL4.50 (Promega, #E1310) or the enhanced green fluorescent protein (EGFP) expression plasmid pEF‐GFP (Addgene, #11154). The pEF‐Luc plasmid used in Figures 1 and S1 was prepared by replacing the EGFP gene in pEF‐GFP with the luc2 gene.…”

Section: Methodsmentioning

confidence: 99%

Nonviral gene delivery to T cells with Lipofectamine LTX

Harris

Zimmerman

Warga

et al. 2021

Biotech & Bioengineering

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Retroviral gene delivery is widely used in T cell therapies for hematological cancers. However, viral vectors are expensive to manufacture, integrate genes in semirandom patterns, and their transduction efficiency varies between patients. In this study, several nonviral gene delivery vehicles, promoters, and additional variables were compared to optimize nonviral transgene delivery and expression in both Jurkat and primary T cells. Transfection of Jurkat cells was maximized to a high efficiency (63.0% ± 10.9% EGFP+ cells) by transfecting cells with Lipofectamine LTX in X‐VIVO 15 media. However, the same method yielded a much lower transfection efficiency in primary T cells (8.1% ± 0.8% EGFP+). Subsequent confocal microscopy revealed that a majority of the lipoplexes did not enter the primary T cells, which might be due to relatively low expression levels of heparan sulfate proteoglycans detected via messenger RNA‐sequencing. Pyrin and HIN (PYHIN) DNA sensors (e.g., AIM2 and IFI16) that can induce apoptosis or repress transcription after binding cytoplasmic DNA were also detected at high levels in primary T cells. Therefore, transfection of primary T cells appears to be limited at the level of cellular uptake or DNA sensing in the cytoplasm. Both of these factors should be considered in the development of future viral and nonviral T cell gene delivery methods.

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The Luc2 gene enhances reliability of bicistronic assays

Abstract: Abbreviations:Ct -threshold cycle; IRES -Internal ribosome entry site; NMD -nonsense-mediated decay; qRT-PCR -quantitative reverse transcription PCR; sORF -short ORF.

Cited by 6 publications

References 20 publications

Bioluminescence Imaging Applications in Cancer: A Comprehensive Review

Bioluminescence Imaging Applications in Cancer: A Comprehensive Review

Improved dynamic monitoring of transcriptional activity during longitudinal analysis in the mouse brain

Nonviral gene delivery to T cells with Lipofectamine LTX

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