Temporally and spatially defined changes in Ca2+ concentration in distinct compartments of cells represent a universal information code in plants. Recently, it has become evident that Ca2+ signals ...not only govern intracellular regulation but also appear to contribute to long distance or even organismic signal propagation and physiological response regulation. Ca2+ signals are shaped by an intimate interplay of channels and transporters, and during past years important contributing individual components have been identified and characterized. Ca2+ signals are translated by an elaborate toolkit of Ca2+-binding proteins, many of which function as Ca2+ sensors, into defined downstream responses. Intriguing progress has been achieved in identifying specific modules that interconnect Ca2+ decoding proteins and protein kinases with downstream target effectors, and in characterizing molecular details of these processes. In this review, we reflect on recent major advances in our understanding of Ca2+ signaling and cover emerging concepts and existing open questions that should be informative also for scientists that are currently entering this field of ever-increasing breath and impact.
Dengue replicons are powerful tools used in studying virus biology as well as in high-throughput screening of drug candidates. Replicon constructs are developed as genomic (consists of all the viral ...protein genes) or sub-genomic (consists of only nonstructural protein genes) and are used to study various aspects of the virus life cycle such as genome replication and virus assembly. In addition, a replicon usually includes a reporter gene used in monitoring virus replication. In this chapter, we provide methods to develop both genomic and sub-genomic dengue replicons with a luciferase reporter and describe different assays to utilize these systems.
The caspase-mediated regulation of many cellular processes, including apoptosis, justifies the substantial interest in understanding all of the biological features of these enzymes. To complement ...functional assays, it is crucial to identify caspase-activating cells in live tissues. Our work describes novel initiator caspase reporters that, for the first time, provide direct information concerning the initial steps of the caspase activation cascade in
tissues. One of our caspase sensors capitalises on the rapid subcellular localisation change of a fluorescent marker to uncover novel cellular apoptotic events relating to the actin-mediated positioning of the nucleus before cell delamination. The other construct benefits from caspase-induced nuclear translocation of a QF transcription factor. This feature enables the genetic manipulation of caspase-activating cells and reveals the spatiotemporal patterns of initiator caspase activity. Collectively, our sensors offer experimental opportunities not available by using previous reporters and have proven useful to illuminate previously unknown aspects of caspase-dependent processes in apoptotic and non-apoptotic cellular scenarios.
STARR-seq — Principles and applications Muerdter, Felix; Boryń, Łukasz M.; Arnold, Cosmas D.
Genomics (San Diego, Calif.),
09/2015, Letnik:
106, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Differential gene expression is the basis for cell type diversity in multicellular organisms and the driving force of development and differentiation. It is achieved by cell type-specific ...transcriptional enhancers, which are genomic DNA sequences that activate the transcription of their target genes. Their identification and characterization is fundamental to our understanding of gene regulation. Features that are associated with enhancer activity, such as regulatory factor binding or histone modifications can predict the location of enhancers. Nonetheless, enhancer activity can only be assessed by transcriptional reporter assays. Over the past years massively parallel reporter assays have been developed for large scale testing of enhancers. In this review we focus on the principles and applications of STARR-seq, a functional assay that quantifies enhancer strengths in complex candidate libraries and thus allows activity-based enhancer identification in entire genomes. We explain how STARR-seq works, discuss current uses and give an outlook to future applications.
Whole-cell and cell-free transcription-translation biosensors have recently become favorable alternatives to conventional detection methods, as they are cost-effective, environmental friendly, and ...easy to use. Importantly, the biological responses from the biosensors need to be converted into a physicochemical signal for easy detection, and a variety of genetic reporters have been employed for this purpose. Reporter gene selection is vital to a sensor performance and application success. However, it was largely based on trial and error with very few systematic side-by-side investigations reported. To address this bottleneck, here we compared eight reporters from three reporter categories, i.e., fluorescent (gfpmut3, deGFP, mCherry, mScarlet-I), colorimetric (lacZ), and bioluminescent (luxCDABE from Aliivibrio fischeri and Photorhabdus luminescens, NanoLuc) reporters, under the control of two representative biosensors for mercury- and quorum-sensing molecules. Both whole-cell and cell-free formats were investigated to assess key sensing features including limit of detection (LOD), input and output dynamic ranges, response time, and output visibility. For both whole-cell biosensors, the lowest detectable concentration of analytes and the fastest responses were achieved with NanoLuc. Notably, we developed, to date, the most sensitive whole-cell mercury biosensor using NanoLuc as reporter, with an LOD ≤ 50.0 fM HgCl2 30 min postinduction. For cell-free biosensors, overall, NanoLuc and deGFP led to shorter response time and lower LOD than the others. This comprehensive profile of diverse reporters in a single setting provides a new important benchmark for reporter selection, aiding the rapid development of whole-cell and cell-free biosensors for various applications in the environment and health.
In the post‐genomic era, thousands of putative noncoding regulatory regions have been identified, such as enhancers, promoters, long noncoding RNAs (lncRNAs), and a cadre of small peptides. These ...ever‐growing catalogs require high‐throughput assays to test their functionality at scale. Massively parallel reporter assays have greatly enhanced the understanding of noncoding DNA elements en masse. Here, we present a massively parallel RNA assay (MPRNA) that can assay 10,000 or more RNA segments for RNA‐based functionality. We applied MPRNA to identify RNA‐based nuclear localization domains harbored in lncRNAs. We examined a pool of 11,969 oligos densely tiling 38 human lncRNAs that were fused to a cytosolic transcript. After cell fractionation and barcode sequencing, we identified 109 unique RNA regions that significantly enriched this cytosolic transcript in the nucleus including a cytosine‐rich motif. These nuclear enrichment sequences are highly conserved and over‐represented in global nuclear fractionation sequencing. Importantly, many of these regions were independently validated by single‐molecule RNA fluorescence in situ hybridization. Overall, we demonstrate the utility of MPRNA for future investigation of RNA‐based functionalities.
Synopsis
A new tiling‐based method maps functional domains in RNAs in a high‐throughput manner, allowing the identification of sequence motifs that contribute to the nuclear enrichment of long non‐coding RNAs.
Massively Parallel RNA Assay (MPRNA) is a universally applicable method to survey RNA‐based functionalities in a high‐throughput manner.
MPRNA identifies 109 nuclear enrichment sequences across 29 of 38 lncRNAs tested.
A C‐rich motif is generally enriched in nuclear versus cytoplasmic transcripts.
RNA‐FISH Fish reveals that large sequence domains are sufficient to localize otherwise cytoplasmic RNA to the nucleus.
MPRNA shows that nuclear lncRNAs have unique and large (˜500 bp) nuclear localization domains
A new tiling‐based method maps functional domains across a panel of long non‐coding RNAs, identifying sequence motifs that retain these RNAs in the nucleus.
Prime editing is a recently developed CRISPR/Cas9 based gene engineering tool that allows the introduction of short insertions, deletions, and substitutions into the genome. However, the efficiency ...of prime editing, which typically achieves editing rates of around 10%-30%, has not matched its versatility. Here, we introduce the prime editor activity reporter (PEAR), a sensitive fluorescent tool for identifying single cells with prime editing activity. PEAR has no background fluorescence and specifically indicates prime editing events. Its design provides apparently unlimited flexibility for sequence variation along the entire length of the spacer sequence, making it uniquely suited for systematic investigation of sequence features that influence prime editing activity. The use of PEAR as an enrichment marker for prime editing can increase the edited population by up to 84%, thus significantly improving the applicability of prime editing for basic research and biotechnological applications.
Royal jelly (RJ) has beneficial effects on human health, and some of these effects are reported to be the result of its estrogenic activity; however, chemicals with estrogenic activities may disrupt ...physiological estrogen signaling leading to adverse effects on human health. Thus, clarification of the mode of action of RJ is needed. Here, we investigated whether the estrogen-like actions of RJ are induced via estrogen receptors (ERs)–mediated genomic actions by using an in vitro reporter assay in human choriocarcinoma JEG3 cells and an estrogen-responsive reporter (E-Rep) mouse line that can be used to sensitively detect transactivation of ERs in multiple organs simultaneously. In the in vitro reporter assay, ERs–dependent transcriptional activity was significantly increased by 17β-estradiol (E2) treatment at concentrations of 1 nM and above, confirming that the assay was highly responsive to estrogen; however, RJ did not exhibit any agonist activity via either the α or β form of ER. Similarly, in E-Rep mice, E2 showed significant ERs–dependent genomic action in 17 tissue types including uterus and mammary gland, whereas RJ did not. Thus, unlike endocrine-disrupting chemicals, the estrogen-like activity of RJ is unlikely to be due to genomic actions via ERs.
For the past several decades, chimeric antigen receptor T-cell therapies have shown promise in the treatment of cancers. These treatments would greatly benefit from companion imaging biomarkers to ...follow the trafficking of T cells in vivo.
Using synthetic biology, we engineered T cells with a chimeric receptor synthetic intramembrane proteolysis receptor (SNIPR) that induces overexpression of an exogenous reporter gene cassette on recognition of specific tumor markers. We then applied a SNIPR-based PET reporter system to 2 cancer-relevant antigens, human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII), commonly expressed in breast and glial tumors, respectively.
Antigen-specific reporter induction of the SNIPR PET T cells was confirmed in vitro using green fluorescent protein fluorescence, luciferase luminescence, and the HSV-TK PET reporter with 9-(4-
F-fluoro-3-hydroxymethylbutyl)guanine (
FFHBG). T cells associated with their target antigens were successfully imaged using PET in dual-xenograft HER2+/HER2- and EGFRvIII+/EGFRvIII- animal models, with more than 10-fold higher
FFHBG signals seen in antigen-expressing tumors versus the corresponding controls.
The main innovation found in this work was PET detection of T cells via specific antigen-induced signals, in contrast to reporter systems relying on constitutive gene expression.
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