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Protein synthesis is influenced by the chemical and structural properties of the amino acids incorporated into the polypeptide chain. Motifs containing consecutive prolines can slow the translation speed and cause ribosome stalling. Translation elongation factor P (EF-P) facilitates peptide bond formation in these motifs, thereby alleviating stalled ribosomes and restoring the regular translational speed. Ribosome pausing at various polyproline motifs has been intensively studied using a range of sophisticated techniques, including ribosome profiling, proteomics, and in vivo screening, with reporters incorporated into the chromosome. However, the full spectrum of motifs that cause translational pausing in Escherichia coli has not yet been identified. Here, we describe a plasmid-based dual reporter for rapid assessment of pausing motifs. This reporter contains two coupled genes encoding mScarlet-I and chloramphenicol acetyltransferase to screen motif libraries based on both bacterial fluorescence and survival. In combination with a diprolyl motif library, we used this reporter to reveal motifs of different pausing strengths in an E. coli strain lacking efp . Subsequently, we used the reporter for a high-throughput screen of four motif libraries, with and without prolines at different positions, sorted by fluorescence-associated cell sorting (FACS) and identify new motifs that influence the translational efficiency of the fluorophore. Our study provides an in vivo platform for rapid screening of amino acid motifs that affect translational efficiencies.

Enzymes that produce volatile metabolites can be coded into genetic circuits to report nondisruptively on microbial behaviors in hard-to-image soils. However, these enzyme reporters remain challenging to apply in gene transfer studies due to leaky off states that can lead to false positives. To overcome this problem, we designed a reporter that uses ribozyme-mediated gene-fragment complementation of a methyl halide transferase (MHT) to regulate the synthesis of methyl halide gases. We split the mht gene into two nonfunctional fragments and attached these to a pair of splicing ribozyme fragments. While the individual mht -ribozyme fragments did not produce methyl halides when transcribed alone in Escherichia coli , coexpression resulted in a spliced transcript that translated the MHT reporter. When cells containing one mht -ribozyme fragment transcribed from a mobile plasmid were mixed with cells that transcribed the second mht -ribozyme fragment, methyl halides were only detected following rare conjugation events. When conjugation was performed in soil, it led to a 16-fold increase in methyl halides in the soil headspace. These findings show how ribozyme-mediated gene-fragment complementation can achieve tight control of protein reporter production, a level of control that will be critical for monitoring the effects of soil conditions on gene transfer and the fidelity of biocontainment measures developed for environmental applications.

Primary hepatocytes are widely utilized for investigating drug efficacy and toxicity, yet variations between batches and limited proliferation capacity present significant challenges. HepaRG cells are versatile cells, capable of maintaining an undifferentiated state and differentiating through dimethyl sulfoxide treatment, allowing for molecular analysis of hepatocyte plasticity. To elucidate the underlying molecular mechanisms of HepaRG cell plasticity, we used CYP3A4G/7R HepaRG cells engineered to express DsRed under the control of the fetus-specific CYP3A7 gene and EGFP under the adult-specific CYP3A4 gene promoter. In time-lapse imaging of CYP3A4G/7R HepaRG cells, we observed CYP3A7-DsRed expression transitioning from negative to positive during proliferation period and CYP3A4-GFP expression activating during differentiation. The de-differentiation potency of differentiated CYP3A4G/7R HepaRG cells was assessed using inhibitors and cytokines. It was found that Y-27632 (Y), A-83-01 (A), and CHIR99021 (C) (collectively referred to as YAC), which are known to promote liver regeneration in mice, did not induce CYP3A7-DsRed expression. Instead, these inhibitors increased CYP3A4-GFP expressing population. Furthermore, CHIR99021 alone increased CYP3A4-GFP-positive cells, while Wnt3a treatment increased CYP3A7-DsRed-positive cells, suggesting that Wnt signaling plays distinct roles in HepaRG cells. It was apparent that de-differentiated cells had increased CYP3A4 activity after a second round of differentiation, compared to differentiated cells after the first round. Transcriptomic analysis of HepaRG cells revealed distinct profiles between proliferative, differentiated, and de-differentiated states, highlighting their robust plasticity. Notably, hepatoblastic cells de-differentiated by YAC or C displayed transcriptome patterns similar to undifferentiated cells, whereas CYP3A7-DsRed and CYP3A4-GFP exhibited expression patterns different from those of undifferentiated cells. These findings underscore the dynamic nature of HepaRG cells while cautioning against solely relying on CYP3 family gene expression as a marker of differentiation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Asano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The massed-spaced effect is a hallmark feature of memory formation. We now demonstrate this effect in two separate non-neural, immortalized cell lines stably expressing a short-lived luciferase reporter controlled by a CREB-dependent promoter. We emulate training using repeated pulses of forskolin and/or phorbol ester, and, as a proxy for memory, measure luciferase expression at various points after training. Four spaced pulses of either agonist elicit stronger and more sustained luciferase expression than a single "massed" pulse. Spaced pulses also result in stronger and more sustained activation of molecular factors critical for memory formation, ERK and CREB, and inhibition of ERK or CREB blocks the massed-spaced effect. Our findings show that canonical features of memory do not necessarily depend on neural circuitry, but can be embedded in the dynamics of signaling cascades conserved across different cell types., (© 2024. The Author(s).)

CRISPR-Cas9 editing triggers activation of the TP53-p21 pathway, but the impacts of different editing components and delivery methods have not been fully explored. In this study, we introduce a p21-mNeonGreen reporter iPSC line to monitor TP53-p21 pathway activation. This reporter enables dynamic tracking of p21 expression via flow cytometry, revealing a strong correlation between p21 expression and indel frequencies, and highlighting its utility in guide RNA screening. Our findings show that p21 activation is significantly more pronounced with double-stranded oligodeoxynucleotides (ODNs) or adeno-associated viral vectors (AAVs) compared to their single-stranded counterparts. Lentiviral vectors (LVs) and integrase-defective lentiviral vectors induce notably lower p21 expression than AAVs, suggesting their suitability for gene therapy in sensitive cells such as hematopoietic stem cells or immune cells. Additionally, specific viral promoters like SFFV significantly amplify p21 activation, emphasizing the critical role of promoter selection in vector development. Thus, the p21-mNeonGreen reporter iPSC line is a valuable tool for assessing the potential adverse effects of gene editing methodologies and vectors. Highlights Established a p21-mNeonGreen reporter iPSC line to track activation of the TP53-p21 pathway. Found a direct correlation between p21-mNeonGreen expression and indel frequencies, aiding in gRNA screening. Showed that LVs are preferable over AAVs for certain cells due to lower p21 activation, with viral promoter choice impacting p21 response., (© The Author(s) 2024. Published by Oxford University Press.)

Cassava ( Manihot esculenta Crantz) is a staple crop in tropical and subtropical regions, valued for its high starch content in roots. Effective genetic transformation and genome editing of cassava require efficient screening methods for transgenic and edited plants. In this study, a visual selection marker system using an R2R3-MYB transcription factor anthocyanin 1 gene ( HbAN1, LOC110667474) from a rubber tree ( Hevea brasiliensis Müll. Arg .) has been developed to facilitate the identification of transgenic cassava plants. Transgenic cassava lines expressing HbAN1 accumulated anthocyanins in their leaves, allowing for easy visual identification without the need for destructive assays or specialized equipment. Importantly, the accumulation of anthocyanins did not affect the regeneration or transformation efficiency of cassava. Additionally, the AR-CRISPR/Cas9-gRNA system with the HbAN1 gene as a marker produced MeCDD4 gene-edited cassava mutants with purple leaves, demonstrating successful editing. This anthocyanin-based visual reporter (AR) system will provide an effective tool for genetic transformation and genome editing in cassava.

Glycogen, a branched polysaccharide organized into glycogen granules (GGs), is delivered from the cytoplasm to the lysosomes of hepatocytes by STBD1-driven selective autophagy (glycophagy). Recently, we developed Komagataella phaffii yeast as a simple model of GG autophagy and found that it proceeds non-selectively under nitrogen starvation conditions. However, another group, using Saccharomyces cerevisiae as a model, found that glycogen is a non-preferred cargo of nitrogen starvation-induced bulk autophagy. To clarify cargo characteristics of K. phaffii GGs, we used the same glycogen synthase-based reporter (Gsy1-GFP) of GG autophagy in K. phaffii as was used in S. cerevisiae . The K. phaffii Gsy1-GFP marked the GGs and reported on their autophagic degradation during nitrogen starvation, as expected. However, unlike in S. cerevisiae , glycogen synthase-marked GGs were delivered to the vacuole and degraded there with the same efficiency as a cytosolic glycogen synthase in glycogen-deficient cells, suggesting that glycogen is a neutral cargo of bulk autophagy in K. phaffii . We verified our findings with a new set of reporters based on the glycogen-binding CBM20 domain of human STBD1. The GFP-CBM20 and mCherry-CBM20 fusion proteins tagged GGs, reported about the autophagy of GGs, and confirmed that GGs in K. phaffii are neither preferred nor non-preferred substrates of bulk autophagy. They are its neutral substrates.

The human malaria parasite Plasmodium falciparum causes the most severe form of malaria in endemic regions and is transmitted via mosquito bites. To better understand the biology of this deadly pathogen, a variety of P. falciparum reporter lines have been generated using transgenic approaches to express reporter proteins, such as fluorescent proteins and luciferases. This review discusses the advances in recently generated P. falciparum transgenic reporter lines, which will aid in the investigation of parasite physiology and the discovery of novel antimalarial drugs. Future prospects for the generation of new and superior human malaria parasite reporter lines are also discussed, and unresolved questions in malaria biology are highlighted to help boost support for the development and implementation of malaria treatments., Competing Interests: Declaration of interests Shinya Miyazaki declares grant/research funding from Shionogi & Co., Ltd., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Nipah virus (NiV) causes near-annual outbreaks of fatal encephalitis and respiratory disease in South Asia with a high mortality rate (∼70%). Since there are no approved therapeutics for NiV disease in humans, the WHO has designated NiV and henipaviral diseases priority pathogens for research and development. We generated a new recombinant green fluorescent reporter NiV of the circulating Bangladesh genotype (rNiV-B-ZsG) and optimized it alongside our previously generated Malaysian genotype reporter counterpart (rNiV-M-ZsG) for antiviral screening in primary-like human respiratory cell types. Validating our platform for rNiV-B-ZsG with a synthetic compound library directed against viral RNA-dependent RNA polymerases, we identified a hit compound and confirmed its sub-micromolar activity against wild-type NiV, green fluorescent reporter, and the newly constructed bioluminescent red fluorescent double reporter (rNiV-B-BREP) NiV. We furthermore demonstrated that rNiV-B-ZsG and rNiV-B-BREP viruses showed pathogenicity comparable to wild-type NiV-B in the Syrian golden hamster model of disease, supporting additional use of these tools for both pathogenesis and advanced pre-clinical studies in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Clostridium butyricum has emerged as a promising candidate for both industrial and medical biotechnologies, underscoring the key pursuit of stable gene overexpression in engineering C. butyricum. Unlike antibiotic-selective vectors, native-cryptic plasmids can be utilized for antibiotic-free expression systems in bacteria but have not been effectively exploited in C. butyricum to date. This study focuses on leveraging these plasmids, pCB101 and pCB102, in C. butyricum DSM10702 for stable gene overexpression without antibiotic selection via efficient gene integration using the SacB-based allelic exchange method. Integration of reporter IFP2.0 and glucuronidase generated sustained near-infrared fluorescence and robust enzyme activity across successive subcultures. Furthermore, successful secretion of a cellulase, Cel9M, and the human interleukin 10 from pCB102 highlights native-cryptic plasmids' potential in conferring stable gene products for industrial and medical applications in C. butyricum. This work appears to be the first study to harness the Clostridium native-cryptic plasmid for stable gene overexpression without antibiotics, thereby advancing the biotechnological prospects of C. butyricum., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Philippe Lambin reports a relationship with Radiomics SA that includes: equity or stocks and funding grants. Philippe Lambin reports a relationship with Convert Pharmaceuticals SA that includes: equity or stocks and funding grants. Philippe Lambin reports a relationship with LivingMed Biotech srl that includes: equity or stocks and funding grants. Philippe Lambin reports a relationship with BHV srl that includes: consulting or advisory and travel reimbursement. Philippe Lambin reports a relationship with Roche that includes: consulting or advisory and travel reimbursement. Philippe Lambin reports a relationship with Comunicare SA that includes: equity or stocks. Philippe Lambin reports a relationship with Bactam srl that includes: equity or stocks. Ludwig J. Dubois reports a relationship with Convert Pharmaceuticals SA that includes: equity or stocks. Ludwig J. Dubois reports a relationship with LivingMed Biotech srl that includes: equity or stocks. Jan Theys reports a relationship with Convert Pharmaceuticals SA that includes: equity or stocks. Yanchao Zhang has patent pending to Maastricht University. Jan Theys has patent pending to Maastricht University. Philippe Lambin has patent pending to Maastricht University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Objective: Human embryonic stem cell (hESC; SC)-derived pancreatic β cells can be used to study diabetes pathologies and develop cell replacement therapies. Although current differentiation protocols yield SCβ cells with varying degrees of maturation, these cells still differ from deceased donor human β cells in several respects. We sought to develop a reporter cell line that could be used to dynamically track SCβ cell functional maturation., Methods: To monitor SCβ cell maturation in vitro, we created an IAPP-2A-mScar and INSULIN-2A-EGFP dual fluorescent reporter (INS 2A-EGFP/+ ;IAPP 2A-mScarlet/+ ) hESC line using CRISPR/Cas9. Pluripotent SC were then differentiated using a 7-stage protocol to islet-like cells. Immunohistochemistry, flow cytometry, qPCR, GSIS and electrophysiology were used to characterise resulting cell populations., Results: We observed robust expression of EGFP and mScarlet fluorescent proteins in insulin- and IAPP-expressing cells without any compromise to their differentiation. We show that the proportion of insulin-producing cells expressing IAPP increases over a 4-week maturation period, and that a subset of insulin-expressing cells remain IAPP-free. Compared to this IAPP-free population, we show these insulin- and IAPP-expressing cells are less polyhormonal, more glucose-sensitive, and exhibit decreased action potential firing in low (2.8 mM) glucose., Conclusions: The INS 2A-EGFP/+ ;IAPP 2A-mScarlet/+ hESC line provides a useful tool for tracking populations of maturing hESC-derived β cells in vitro. This tool has already been shared with 3 groups and is freely available to all., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Measurement of gene expression in the brain requires invasive analysis of brain tissue or non-invasive methods that are limited by low sensitivity. Here we introduce a method for non-invasive, multiplexed, site-specific monitoring of endogenous gene or transgene expression in the brain through engineered reporters called released markers of activity (RMAs). RMAs consist of an easily detectable reporter and a receptor-binding domain that enables transcytosis across the brain endothelium. RMAs are expressed in the brain but exit into the blood, where they can be easily measured. We show that expressing RMAs at a single mouse brain site representing approximately 1% of the brain volume provides up to a 100,000-fold signal increase over the baseline. Expression of RMAs in tens to hundreds of neurons is sufficient for their reliable detection. We demonstrate that chemogenetic activation of cells expressing Fos-responsive RMA increases serum RMA levels >6-fold compared to non-activated controls. RMAs provide a non-invasive method for repeatable, multiplexed monitoring of gene expression in the intact animal brain., Competing Interests: Competing interests J.O.S. and S.L. are co-inventors on a US patent application that incorporates discoveries described in this paper. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

One of the major processes occurring during the healing of a fractured long bone is chondrogenesis, leading to the formation of the soft callus, which subsequently undergoes endochondral ossification and ultimately bridges the fracture site. Thus, understanding the molecular mechanisms of chondrogenesis can enhance our knowledge of the fracture repair process. One such molecular process is calciun (Ca ++ ) signaling, which is known to play a critical role in the development and regeneration of multiple tissues, including bone, in response to external stimuli. Despite the existence of various mouse models for studying Ca ++ signaling, none of them were designed to specifically examine the skeletal system or the various musculoskeletal cell types. As such, we generated a genetically engineered mouse model that is specific to cartilage (crossed with Col2a1 Cre mice) to study chondrocytes. Herein, we report on the characterization of this transgenic mouse line using conditional expression of GCaMP6f, a Ca ++ -indicator protein. Specifically, this mouse line exhibits increased GCaMP6f fluorescence following Ca ++ binding in chondrocytes. Using this model, we show real-time Ca ++ signaling in embryos, newborn and adult mice, as well as in fracture calluses. Further, robust expression of GCaMP6f in chondrocytes can be easily detected in embryos, neonates, adults, and fracture callus tissue sections. Finally, we also report on Ca ++ signaling pathway gene expression, as well as real-time Ca ++ transient measurements in fracture callus chondrocytes. Taken together, these mice provide a new experimental tool to study chondrocyte-specific Ca ++ signaling during skeletal development and regeneration, as well as various in vitro perturbations., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cell cycle regulation is critical to blood vessel formation and function, but how the endothelial cell cycle integrates with vascular regulation is not well-understood, and available dynamic cell cycle reporters do not precisely distinguish all cell cycle stage transitions in vivo. Here we characterized a recently developed improved cell cycle reporter (PIP-FUCCI) that precisely delineates S phase and the S/G2 transition. Live image analysis of primary endothelial cells revealed predicted temporal changes and well-defined stage transitions. A new inducible mouse cell cycle reporter allele was selectively expressed in postnatal retinal endothelial cells upon Cre-mediated activation and predicted endothelial cell cycle status. We developed a semi-automated zonation program to define endothelial cell cycle status in spatially defined and developmentally distinct retinal areas and found predicted cell cycle stage differences in arteries, veins, and remodeled and angiogenic capillaries. Surprisingly, the predicted dearth of S-phase proliferative tip cells relative to stalk cells at the vascular front was accompanied by an unexpected enrichment for endothelial tip and stalk cells in G2, suggesting G2 stalling as a contribution to tip-cell arrest and dynamics at the front. Thus, this improved reporter precisely defines endothelial cell cycle status in vivo and reveals novel G2 regulation that may contribute to unique aspects of blood vessel network expansion., Competing Interests: Declarations Competing interest The authors declare no competing interests., (© 2024. The Author(s).)

During development, most cells experience a progressive restriction of fate that ultimately results in a fully differentiated mature state. Understanding more about the gene expression patterns that underlie developmental programs can inform engineering efforts for new or optimized forms. Here, we present a four-state integrase-based recorder of gene expression history and demonstrate its use in tracking gene expression events in Arabidopsis thaliana in two developmental contexts: lateral root initiation and stomatal differentiation. The recorder uses two serine integrases to mediate sequential DNA recombination events, resulting in step-wise, history-dependent switching between expression of fluorescent reporters. By using promoters that express at different times along each of the two differentiation pathways to drive integrase expression, we tie fluorescent status to an ordered progression of gene expression along the developmental trajectory. In one snapshot of a mature tissue, our recorder is able to reveal past gene expression with single cell resolution. In this way, we are able to capture heterogeneity in stomatal development, confirming the existence of two alternate paths of differentiation., (© 2024. The Author(s).)

Neuromuscular signal transmission is affected in various diseases including myasthenia gravis, congenital myasthenic syndromes, and sarcopenia. We used an ATF2-luciferase system to monitor the phosphorylation of MuSK in HEK293 cells introduced with MUSK and LRP4 cDNAs to find novel chemical compounds that enhanced agrin-mediated acetylcholine receptor (AChR) clustering. Four compounds with similar chemical structures carrying benzene rings and heterocyclic rings increased the luciferase activities 8- to 30-folds, and two of them showed continuously graded dose dependence. The effects were higher than that of disulfiram, a clinically available aldehyde dehydrogenase inhibitor, which we identified to be the most competent preapproved drug to enhance ATF2-luciferase activity in the same assay system. In C2C12 myotubes, all the compounds increased the area, intensity, length, and number of AChR clusters. Three of the four compounds increased the phosphorylation of MuSK, but not of Dok7, JNK. ERK, or p38. Monitoring cell toxicity using the neurite elongation of NSC34 neuronal cells as a surrogate marker showed that all the compounds had no effects on the neurite elongation up to 1 μM. Extensive docking simulation and binding structure prediction of the four compounds with all available human proteins using AutoDock Vina and DiffDock showed that the four compounds were unlikely to directly bind to MuSK or Dok7, and the exact target remained unknown. The identified compounds are expected to serve as a seed to develop a novel therapeutic agent to treat defective NMJ signal transmission., Competing Interests: Declaration of competing interest The authors declare that none of the authors have conflict of interest in this manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Genes are often regulated by multiple enhancers. It is poorly understood how the individual enhancer activities are combined to control promoter activity. Anecdotal evidence has shown that enhancers can combine sub-additively, additively, synergistically, or redundantly. However, it is not clear which of these modes are more frequent in mammalian genomes. Here, we systematically tested how pairs of enhancers activate promoters using a three-way combinatorial reporter assay in mouse embryonic stem cells. By assaying about 69,000 enhancer-enhancer-promoter combinations we found that enhancer pairs generally combine near-additively. This behaviour was conserved across seven developmental promoters tested. Surprisingly, these promoters scale the enhancer signals in a non-linear manner that depends on promoter strength. A housekeeping promoter showed an overall different response to enhancer pairs, and a smaller dynamic range. Thus, our data indicate that enhancers mostly act additively, but promoters transform their collective effect non-linearly., Competing Interests: MM, Bv No competing interests declared, FC co-founder of enGene Statistics GmbH, (© 2023, Martinez-Ara et al.)

The crustacean Daphnia magna is an emerging model for ecological and toxicological genomics. However, the lack of methods for spatial and temporal control of gene expression has impaired the elucidation of molecular mechanisms underlying responses to environments in vivo. Here we report local activation of the hsp70 promoter-driven gene cassette in D. magna by the infrared laser-evoked gene operator (IR-LEGO), a method for heating the target cells with infrared irradiation. We identified the heat-inducible promoter upstream of the D. magna hsp70-A gene. Using this promoter, we generated a transgenic Daphnia harboring the heat-shock responsive GFP reporter gene and confirmed that the GFP gene responds to heat treatment not only in juveniles and adults but also in embryos. We collected embryos from the reporter line and irradiated four different regions of interest in the embryos: a proximal region of the third thoracic segment, a part of the midline, a second maxilla, and a distal region of the endopodite of the second antenna, all of which increased GFP fluorescence with an infrared laser. Our results suggest that the IR-LEGO method is useful for spatial and temporal control of gene expression and would advance the functional genomics in D. magna., (© 2024. The Author(s).)

The development of human induced pluripotent stem cell (iPSC)-based regenerative therapies is challenged by the lack of specific cell markers to isolate differentiated cell types and improve differentiation protocols. This issue is particularly critical for notochordal-like cells and chondrocytes, which are crucial in treating back pain and osteoarthritis, respectively. Both cell types produce abundant proteoglycan aggrecan (ACAN), crucial for the extracellular matrix. We generated two human iPSC lines containing an ACAN-2A-mScarlet reporter. The reporter cell lines were validated using CRISPR-mediated transactivation and functionally validated during notochord and cartilage differentiation. The ability to isolate differentiated cell populations producing ACAN enables their enrichment even in the absence of specific cell markers and allows for comprehensive studies and protocol refinement. ACAN's prevalence in various tissues (e.g., cardiac and cerebral) underscores the reporter's versatility as a valuable tool for tracking matrix protein production in diverse cell types, benefiting developmental biology, matrix pathophysiology, and regenerative medicine.

Gene expression is regulated by transcription factors binding to cis-elements in promoters. However, efficient cis-elements for genetic engineering are rarely reported. In this study, we identified an 11 bp cis-element in the PtoCP1 promoter that drives strong constitutive gene expression in Populus tomentosa . A 2,270 bp promoter region upstream of the PtoCP1 gene's translation start site was cloned and named ProPtoCP1. This promoter controls GUS reporter gene expression in the roots, leaves, and stems of Arabidopsis seedlings. Based on the location and density of cis-elements , the PtoCP1 promoter was divided into four fragments by 5'-end deletions. GUS staining and RT-qPCR revealed a key cis-element at -466 to -441 bp essential for gene expression. Further analysis showed that the MYB-TGACG cis-element is a positive regulator, whereas neither MYB nor TGACG alone drove gene expression. This study enhances our understanding of gene expression regulation by cis-elements and provides a valuable tool for genetic engineering., Competing Interests: The authors declare that they have no competing interests., (© 2024 Peng et al.)

Various techniques using fluorescent reporter probes have been developed, such as GFP transgenic mouse lines that are used to detect spatial-temporal expression levels of genes. Although GFP expression is largely considered non-toxic, recent reports have indicated that under certain conditions GFP can display cellular toxicity. We hereby report the nuclear toxicity of H2B-GFP using a K14 specific Tet-on reporter mouse system. Using this system, GFP accumulates in the nucleus of all K14 expressing cells, such as the ocular surface epithelia and ocular adnexa. Expression of high levels of nuclear GFP during embryonic stages led to an eye open-at-birth (EOB) phenotype and abnormal ocular adnexa development and during adult and aging stages showed notable toxicity to ocular tissues. Other tissues, such as skin, also presented multiple defects associated with H2B-GFP expression. This toxicity was found to be concentration dependent, with homozygous mice presenting extremely high toxicity, while heterozygous mice presented limited toxicity. Upon induction, the accumulation of H2B-GFP in the nucleus of homozygous mice led to apoptosis within 2 weeks. This study therefore shows that although the use of nuclear GFP reporter mice is a valuable tool, at high levels, nuclear GFP can be toxic, leading to cell death and affecting tissue function., (© 2024. The Author(s).)

Cell-free transcription-translation (TXTL) systems expressing genes from linear dsDNA enable the rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present reporter systems based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in E. coli TXTL. The 135 bp GFP10-11 fragment produces a fluorescent signal comparable to its full-length GFP counterpart when reassembling with its complementary protein synthesized from the 535 bp fragment expressed in TXTL. We show that split reporters can be used to characterize promoter libraries, with data qualitatively comparable to full-length GFP and matching in vivo expression measurements. We also use split reporters as small fusion tags to measure the TXTL protein and peptide production yield. Finally, we generalize our concept by providing a luminescent split reporter based on split-nanoluciferase. The ∼80% gene sequence length reduction afforded by split reporters lowers synthesis costs and liberates space for testing larger devices while producing a reliable output. In the peptide production context, the small size of split reporters compared with full-length GFP is less likely to bias peptide solubility assays. We anticipate that split reporters will facilitate rapid and cost-efficient genetic device prototyping, protein production, and interaction assays.

Enhancers are central for the regulation of metazoan transcription but have proven difficult to study, primarily due to a myriad of interdependent variables shaping their activity. Consequently, synthetic biology has emerged as the main approach for dissecting mechanisms of enhancer function. We start by reviewing simple but highly parallel reporter assays, which have been successful in quantifying the complexity of the activator/coactivator mechanisms at enhancers. We then describe studies that examine how enhancers function in the genomic context and in combination with other enhancers, revealing that they activate genes through a variety of different mechanisms, working together as a system. Here, we primarily focus on synthetic reporter genes that can quantify the dynamics of enhancer biology through time. We end by considering the consequences of having many genes and enhancers within a 'local environment', which we believe leads to correlated gene expression and likely reports on the general principles of enhancer biology.

The CRISPR system finds extensive application in molecular biology, but its continuous activity can yield adverse effects. Leveraging programmable CRISPR/Cas9 function via nano-device mediation effectively mitigates these drawbacks. The integration of RNA-sensing platforms into CRISPR thus empowers it as a potent tool for processing internal cell data and modulating gene activity. Here, an intelligent guide RNA-a cis-repressed gRNA synthetic circuit enabling efficient recognition of specific trigger RNAs-is developed. This platform carries two toehold switches and includes an inhibited CrRNA sequence. In this system, the presence of cognate trigger RNA promotes precise binding to the first toehold site, initiating a cascade that releases CrRNA to target a reporter gene (luciferase) in this study. Decoupling the CrRNA segment from the trigger RNA enhances the potential of this genetic logic circuit to respond to specific cellular circumstances, offering promise as a synthetic biology platform., (© 2024. The Author(s).)

Internal ribosomal entry sites (IRESs) recruit the ribosome to promote translation, typically in an m7G cap-independent manner. Although IRESs are well-documented in viral genomes, they have also been reported in mammalian transcriptomes, where they have been proposed to mediate cap-independent translation of mRNAs. However, subsequent studies have challenged the idea of these "cellular" IRESs. Current methods for screening and discovering IRES activity rely on a bicistronic reporter assay, which is prone to producing false positive signals if the putative IRES sequence has a cryptic promoter or cryptic splicing sites. Here, we report an assay for screening IRES activity using a genetically encoded circular RNA comprising a split nanoluciferase (nLuc) reporter. The circular split nLuc reporter is less susceptible to the various sources of false positives that adversely affect the bicistronic IRES reporter assay and provides a streamlined method for screening IRES activity. Using the circular split nLuc reporter, we find that nine reported cellular IRESs have minimal IRES activity. Overall, the circular split nLuc reporter offers a simplified approach for identifying and validating IRESs and exhibits reduced propensity for producing the types of false positives that can occur with the bicistronic reporter assay., (© 2024 Unti et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Reporter alleles are essential for advancing research with human induced pluripotent stem cells (hiPSCs), notably in developmental biology and disease modeling. This study investigates the state-of-the-art gene-editing techniques tailored for generating reporter alleles in hiPSCs, emphasizing their effectiveness in investigating cellular dynamics and disease mechanisms. Various methodologies, including the application of CRISPR/Cas9 technology, are discussed for accurately integrating reporter genes into the specific genomic loci. The synthesis of findings from the studies utilizing these reporter alleles reveals insights into developmental processes, genetic disorder modeling, and therapeutic screening, consolidating the existing knowledge. These hiPSC-derived models demonstrate remarkable versatility in replicating human diseases and evaluating drug efficacy, thereby accelerating translational research. Furthermore, this review addresses challenges and future directions in refining the reporter allele design and application to bolster their reliability and relevance in biomedical research. Overall, this investigation offers a comprehensive perspective on the methodologies, applications, and implications of reporter alleles in hiPSC-based studies, underscoring their essential role in advancing both fundamental scientific understanding and clinical practice.

One problem that has hampered the use of red fluorescent proteins in the fast-developing nematode Caenorhabditis elegans has been the substantial time delay in maturation of several generations of red fluorophores. The recently described mScarlet-I3 protein has properties that may overcome this limitation. We compare here the brightness and onset of expression of CRISPR/Cas9 genome-engineered mScarlet, mScarlet3, mScarlet-I3, and GFP reporter knock-ins. Comparing the onset and brightness of expression of reporter alleles of C. elegans golg-4, encoding a broadly expressed Golgi resident protein, we found that the onset of detection of mScarlet-I3 in the embryo is several hours earlier than older versions of mScarlet and comparable to GFP. These findings were further supported by comparing mScarlet-I3 and GFP reporter alleles for pks-1, a gene expressed in the CAN neuron and cells of the alimentary system, as well as reporter alleles for the pan-neuronal, nuclear marker unc-75. Hence, the relative properties of mScarlet-I3 and GFP do not depend on cellular or subcellular context. In all cases, mScarlet-I3 reporters also show improved signal-to-noise ratio compared to GFP., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Mycobacterium abscessus (Mab) is an emerging pathogen that poses a severe health threat, especially in people with cystic fibrosis and other chronic lung diseases. Available drugs are largely ineffective due to an exquisite intrinsic resistance, making Mab infections only comparable to multidrug-resistant tuberculosis. Current treatment is based on lengthy multidrug therapy, complicated by poor outcomes and high rates of treatment failure, recurrence, and mortality. Thus, finding new and more efficient drugs to combat this pathogen is urgent. However, drug discovery efforts targeting Mab have been limited, and traditional drug screening methods are labor-intensive, low-throughput, and do not reflect clinical effectiveness. Therefore, this work aimed to develop a new, efficient, and reliable tool for drug screening against Mab that can be used in vitro for identifying hits in a high-throughput manner and in vivo to select drug candidates for future clinical trials. We engineered two stable double-reporter strains of Mab capable of emitting strong fluorescent and luminescent signals. This is due to the expression of mScarlet protein and luciferase enzyme or the entire lux operon. Importantly, these strains maintain the same ground characteristics as the non-transformed Mab strain. We show that these new strains can be applied to various setups, from MIC determination in broth cultures and macrophage infection assays to in vivo infection (using the Galleria mellonella model). Using these strains enhances the potential for high-throughput screening of thousands of compounds in a fast and reliable way., Importance: Mycobacterium abscessus (Mab) is currently considered an "incurable nightmare." Its intrinsic resistance, high toxicity, long duration, and low cure rates of available therapies often lead to the clinical decision not to treat. Moreover, one of the significant drawbacks of anti-Mab drug development is the lack of correlation between in vitro susceptibility and clinical efficacy. Most drug screening assays are performed on Mab growing in liquid cultures. But being an intracellular pathogen, inducing granulomas and biofilm formation, the broth culture is far from ideal as in vitro drug-testing setup. This study presents new double-reporter Mab strains that allow direct real-time bacterial detection and quantification in a non-invasive way. These strains can be applied to an extensive range of experimental settings, far surpassing the utility of single-reporter bacteria. They can be used in all steps of the pre-clinical anti-Mab drug development pipeline, constituting a highly valuable tool to increase its success., Competing Interests: The authors declare no conflict of interest.

The dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi) gene regulation technique requires two components: a catalytically inactive Cas9 protein (dCas9) and a single-guide RNA that targets the gene of interest. This system is commonly activated by expressing dCas9 through an inducible gene promoter, but these inducers may affect cellular physiology, and accessibility and permeability of the inducer are limited in relevant model systems. Here, we have developed an alternative approach for CRISPRi activation in the clinical isolate Staphylococcus aureus USA300 LAC, where dCas9 was expressed through endogenous virulence gene promoters (vgp); coagulase, autolysin, or fibronectin-binding protein A. Additionally, we integrated a fluorescent reporter gene into the vgp-CRISPRi system to monitor the activity of the dcas9 -controlling promoter. Testing the efficacy of vgp-CRISPRi by inducing growth arrest (when targeting penicillin-binding protein 1), downregulating target gene expression, or blocking coagulase-dependent coagulation of blood plasma, we provide a proof-of-concept demonstration that the virulence gene promoter-driven CRISPRi system is functional in S. aureus .IMPORTANCEThe presented inducer-free, endogenous virulence gene promoter-induced, dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi system addresses several shortcomings related to the use of inducer-dependent systems such as effects on cell physiology or limitations in permeability, and it avoids the high, putatively toxic levels of dCas9 in CRISPRi systems controlled by strong, constitutive promoters., Competing Interests: The authors declare no conflict of interest.

The development of therapies to combat neurodegenerative diseases is widely recognized as a research priority. Despite recent advances in understanding their molecular basis, there is a lack of suitable early biomarkers to test selected compounds and accelerate their translation to clinical trials. We have investigated the utility of in vivo reporters of cytoprotective pathways (e.g. NRF2, p53) as surrogate early biomarkers of the ALS degenerative disease progression. We hypothesized that cellular stress observed in a model of ALS may precede overt cellular damage and could activate our cytoprotective pathway reporters. To test this hypothesis, we generated novel ALS-reporter mice by crossing the hTDP-43tg model into our oxidative stress/inflammation (Hmox1; NRF2 pathway) and DNA damage (p21; p53 pathway) stress reporter models. Histological analysis of reporter expression in a homozygous hTDP-43tg background demonstrated a time-dependent and tissue-specific activation of the reporters in tissues directly associated with ALS, before moderate clinical signs are observed. Further work is warranted to determine the specific mechanisms by which TDP-43 accumulation leads to reporter activation and whether therapeutic intervention modulates reporters' expression. We anticipate the reporter strategy could be of great value in developing treatments for a range of degenerative disorders.

Studying influenza A viruses (IAVs) requires secondary experimental procedures to detect the presence of the virus in infected cells or animals. The ability to generate recombinant (r)IAV using reverse genetics techniques has allowed investigators to generate viruses expressing foreign genes, including fluorescent and luciferase proteins. These rIAVs expressing reporter genes have allowed for easily tracking viral infections in cultured cells and animal models of infection without the need for secondary approaches, representing an excellent option to study different aspects in the biology of IAV where expression of reporter genes can be used as a readout of viral replication and spread. Likewise, these reporter-expressing rIAVs provide an excellent opportunity for the rapid identification and characterization of prophylactic and/or therapeutic approaches. To date, rIAV expressing reporter genes from different viral segments have been described in the literature. Among those, rIAV expressing reporter genes from the viral NS segment have been shown to represent an excellent option to track IAV infection in vitro and in vivo, eliminating the need for secondary approaches to identify the presence of the virus. Here, we summarize the status on rIAV expressing traceable reporter genes from the viral NS segment and their applications for in vitro and in vivo influenza research.

Estrogen receptor alpha (ERα) is a nuclear receptor that is expressed mainly in the breast, uterus, and ovary, among several other organs. ERα plays important roles in reproduction, mammary gland formation, and glucose homeostasis. Disruption of ERα may result in adverse outcomes, such as cancer, impaired fertility, and abnormal fetal growth. Therefore, identifying compounds that modulate ERα is of great interest due to their potential-endocrine disrupting capability and pharmaceutical applications. To rapidly test tens of thousands of compounds, high-throughput screening assays are essential. Here, we describe high-throughput screening methods, including plating and treatment of cells in 384-well and 1536-well plates and analysis of the resulting data. The two cell lines used, MCF7-VM7Luc4E2 and HEK293-ERα-bla, have been described previously. MCF7-VM7Luc4E2 cells are a stable luciferase reporter gene cell line expressing full-length endogenous estrogen receptor in the MCF7 cell line background, and HEK293-ERα-bla cells stably express an ERα ligand-binding domain/GAL4 DNA-binding domain fusion regulating a UAS β-lactamase reporter gene. These cell lines can be used to identify and confirm ERα modulators. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of a high-throughput ERα reporter gene assay with luminescence readout to identify activators and inhibitors of estrogen receptor α Basic Protocol 2: Use of an orthogonal assay with fluorescence readout to confirm potential estrogen receptor activators or inhibitors., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC.)

Osteoporosis is a major threat to the elderly worldwide. The Wnt signaling pathway plays a critical role in bone development and homeostasis. Sclerostin, a Wnt ligand inhibitor, competes with Wnt ligands for low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) on osteoblasts, thereby suppressing bone formation. Sclerostin-neutralizing monoclonal antibodies (mAbs) have emerged as a potential bone-forming therapy for osteoporosis. A cell-based bioassay which determines the relative activity of a product, related to its mechanism of action, is of great importance from drug discovery to quality control and batch release. Currently used cell-based bioassays for sclerostin-neutralizing mAbs usually use Wnt1 or Wnt3a to stimulate the Wnt pathway; sclerostin is a direct inhibitor of Wnt1 but not Wnt3a. Wnt1 is a highly hydrophobic protein that binds to the producing cell membrane and acts in a juxtacrine manner to stimulate the Wnt pathway in neighboring cells. Bioassays for drugs that induce Wnt1 signaling should be performed in a juxtacrine manner. Here, we present a mechanism of action-reflective, dual cell-based reporter gene assay. In this assay, Wnt1 producer cells are co-cultured with cells containing the Wnt reporter genes, Wnt1 on the producer cells activates the Wnt signaling pathway in the reporter cells that are in direct cell-to-cell contact, and sclerostin-neutralizing mAbs specifically and effectively antagonize the sclerostin-mediated Wnt reporter gene suppression. This bioassay demonstrates good specificity, accuracy, linearity, and precision and is suitable for quality control, stability testing, batch release, and biosimilarity assessment of sclerostin-neutralizing mAbs., Competing Interests: Conflicts of Interest All authors except for Yongjie Lai are employees of Zhuhai United Biopharma Co., Ltd or Zhuhai United Laboratories Co., Ltd. The authors are aware of no affiliations, memberships, funding, or financial interests that might be perceived to affect the objectivity of this work., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The clinical treatment of hepatocellular carcinoma (HCC) is still a heavy burden worldwide. Intracellular microRNAs (miRNAs) commonly express abnormally in cancers, thus they are potential therapeutic targets for cancer treatment. miR-21 is upregulated in HCC whereas miR-122 is enriched in normal hepatocyte but downregulated in HCC. In our study, we first generated a reporter genetic switch compromising of miR-21 and miR-122 sponges as sensor, green fluorescent protein (GFP) as reporter gene and L7Ae:K-turn as regulatory element. The reporter expression was turned up in miR-21 enriched environment while turned down in miR-122 enriched environment, indicating that the reporter switch is able to respond distinctly to different miRNA environment. Furthermore, an AAT promoter, which is hepatocyte-specific, is applied to increase the specificity to hepatocyte. A killing switch with AAT promoter and an apoptosis-inducing element, Bax, in addition to miR-21 and miR-122 significantly inhibited cell viability in Huh-7 by 70 % and in HepG2 by 60 %. By contrast, cell viability was not affected in five non-HCC cells. Thus, we provide a novel feasible strategy to improve the safety of miRNA-based therapeutic agent to cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The activation of yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ) has been implicated in both regeneration and tumorigenesis, thus representing a double-edged sword in tissue homeostasis. However, how the activity of YAP1/TAZ is regulated or what leads to its dysregulation in these processes remains unknown. To explore the upstream stimuli modulating the cellular activity of YAP1/TAZ, we developed a highly sensitive YAP1/TAZ/TEAD-responsive DNA element (YRE) and incorporated it into a lentivirus-based reporter cell system to allow for sensitive and specific monitoring of the endogenous activity of YAP1/TAZ in terms of luciferase activity in vitro and Venus fluorescence in vivo. Furthermore, by replacing YRE with TCF- and NF-κB-binding DNA elements, we demonstrated the applicability of this reporter system to other pathways such as Wnt/β-catenin/TCF- and IL-1β/NF-κB-mediated signaling, respectively. The practicality of this system was evaluated by performing cell-based reporter screening of a chemical compound library consisting of 364 known inhibitors, using reporter-introduced cells capable of quantifying YAP1/TAZ- and β-catenin-mediated transcription activities, which led to the identification of multiple inhibitors, including previously known as well as novel modulators of these signaling pathways. We further confirmed that novel YAP1/TAZ modulators, such as potassium ionophores, Janus kinase inhibitors, platelet-derived growth factor receptor inhibitors, and genotoxic stress inducers, alter the protein level or phosphorylation of endogenous YAP1/TAZ and the expression of their target genes. Thus, this reporter system provides a powerful tool to monitor endogenous signaling activities of interest (even in living cells) and search for modulators in various cellular contexts., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Basal cells are adult stem cells in the airway epithelium and regenerate differentiated cell populations, including the mucosecretory and ciliated cells that enact mucociliary clearance. Human basal cells can proliferate and produce differentiated epithelium in vitro. However, studies of airway epithelial differentiation mostly rely on immunohistochemical or immunofluorescence-based staining approaches, meaning that a dynamic approach is lacking, and quantitative data are limited. Here, we use a lentiviral reporter gene approach to transduce primary human basal cells with bioluminescence reporter constructs to monitor airway epithelial differentiation longitudinally. We generated three constructs driven by promoter sequences from the TP63 , MUC5AC , and FOXJ1 genes to quantitatively assess basal cell, mucosecretory cell, and ciliated cell abundance, respectively. We validated these constructs by tracking differentiation of basal cells in air-liquid interface and organoid ("bronchosphere") cultures. Transduced cells also responded appropriately to stimulation with interleukin 13 (IL-13; to increase mucosecretory differentiation and mucus production) and IL-6 (to increase ciliated cell differentiation). These constructs represent a new tool for monitoring airway epithelial cell differentiation in primary epithelial and/or induced pluripotent stem cell (iPSC)-derived cell cultures. NEW & NOTEWORTHY Orr et al. generated and validated new lentiviral vectors to monitor the differentiation of airway basal cells, goblet cells, or multiciliated cells using bioluminescence.

Objective: Evaluation of Nepeta cataria as a host with specific endogenous metabolite background for transient expression and metabolic engineering of secondary biosynthetic sequences., Results: The reporter gene gfp::licBM3 as well as three biosynthetic genes leading to the formation of the cannabinoid precursor olivetolic acid were adopted to the modular cloning standard GoldenBraid, transiently expressed in two chemotypes of N. cataria and compared to Nicotiana benthamiana. To estimate the expression efficiency in both hosts, quantification of the reporter activity was carried out with a sensitive and specific lichenase assay. While N. benthamiana exhibited lichenase activity of 676 ± 94 μmol g -1  s -1 , N. cataria cultivar '1000', and the cultivar 'Citriodora' showed an activity of 37 ± 8 μmol g -1  s -1 and 18 ± 4 μmol g -1  s -1 , respectively. Further, combinatorial expression of genes involved in cannabinoid biosynthetic pathway acyl-activating enzyme 1 (aae1), olivetol synthase (ols) and olivetolic acid cyclase (oac) in N. cataria cv. resulted presumably in the in vivo production of olivetolic acid glycosides., Conclusion: Nepeta cataria is amenable to Agrobacterium-mediated transient expression and could serve as a novel chassis for the engineering of secondary metabolic pathways and transient evaluation of heterologous genes., (© 2024. The Author(s).)

Getah virus (GETV) is a re-emerging mosquito-borne RNA virus that induces fever, hind limb edema, swollen submandibular lymph nodes, and urticaria in horses. In pigs, the virus often results in stillbirths among pregnant sows, and neurological symptoms leading to death in piglets. Currently, there are no specific treatments or drugs available for GETV infection. The use of reporter viruses to monitor viral replication and spread in real-time within infected cells and animals provides a powerful tool for targeting antiviral drugs throughout the viral life cycle. Their fluorescence-tracked characteristics greatly facilitate virus neutralization tests (VNTs). In this study, we engineered two recombinant viruses by inserting different reporter protein genes at the 3' end of the structural protein gene, an unreported location that can accommodate exogenous genes. The rGEEiLOV and rGEEGFP viruses demonstrated genetic stability for at least five passages and replicated at a rate similar to that of the parental virus in BHK-21 cells. The rGEEGFP virus facilitated viral neutralization testing. Additionally, we used the reporter virus rGEEGFP to confirm ivermectin, a broad-spectrum antiparasitic agent, as a potential inhibitor of GETV in vitro. Ivermectin appears to inhibit the early replication stages of the virus and can block cell-to-cell viral transmission. In conclusion, rGEEGFP holds significant potential for antiviral screening to identify specific inhibitors against GETV and for use in viral neutralization tests., Competing Interests: Declaration of competing interest The research was conducted in the absence of any commercial or financial relationships., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We genetically manipulated HaCaT cells, a spontaneously immortalised normal keratinocyte cell line, to stably express two different coloured luciferase reporter genes, driven by interleukin 8 (IL-8) and ubiquitin-C (UBC) promoters, respectively. Subsequently, we generated a three-dimensional (3D) skin-like in vitro composite (SLIC) utilising these cells, with the objective of monitoring bioluminescence emitted from the SLIC. This SLIC was generated on non-woven silica fibre membranes in differentiation medium. Immunohistochemical analyses of skin differentiation markers in the SLIC revealed the expression of keratins 2 and 10, filaggrin, and involucrin, indicating mature skin characteristics. This engineered SLIC was employed for real-time bioluminescence monitoring, allowing the assessment of time- and dose-dependent responses to UV stress, as well as to hydrophilic and hydrophobic chemical loads. Notably, evaluation of responses to hydrophobic substances has been challenging with conventional 2D cell culture methods, suggesting the need for a new approach, which this technology could address. Our observations suggest that engineered SLIC with constitutively expressing reporters driven by selected promoters which are tailored to specific objectives, significantly facilitates assays exploring the physiological functions of skin cells based on genetic response mechanisms. It also highlights new avenues for evaluating the physiological impacts of various compounds designed for topical application to human skin., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Competing Interests: Conflict of interest statement. None declared.

Investigating how transcription factors control complex cellular processes requires tools that enable responses to be visualised at the single-cell level and their cell fate to be followed over time. For example, the tumour suppressor p53 (also called TP53 in humans and TRP53 in mice) can initiate diverse cellular responses by transcriptional activation of its target genes: Puma to induce apoptotic cell death and p21 to induce cell cycle arrest/cell senescence. However, it is not known how these processes are regulated and initiated in different cell types. Also, the context-dependent interaction partners and binding loci of p53 remain largely elusive. To be able to examine these questions, we here developed knock-in mice expressing triple-FLAG-tagged p53 to facilitate p53 pull-down and two p53 response reporter mice, knocking tdTomato and GFP into the Puma/Bbc3 and p21 gene loci, respectively. By crossing these reporter mice into a p53-deficient background, we show that the new reporters reliably inform on p53-dependent and p53-independent initiation of both apoptotic or cell cycle arrest/senescence programs, respectively, in vitro and in vivo., (© 2024. The Author(s).)

Various reporter genes have been developed to study gene expression pattern and gene regulation. The RUBY reporter gene was recently developed and widely used, because of its visible and noninvasive advantages. However, quantitative analysis of RUBY gene expression levels was lacking. In this study, we introduce a novel betalain quantification method in combination with the tobacco transient expression system. The betalain produced in tobacco leaves was extracted and purified, and its concentration was quantitatively measured. We successfully applied this approach in studying the transcriptional regulation of ARC5 gene by transcription factors CPD25 and CPD45. Furthermore, with this method, we showed that the gene expression of RCA and Rbcs1A gene were regulated by light, transcription factors HY5 and PIFs through G-box and I-box elements. The development of this betalain quantification approach with the tobacco transient expression system offers a cost-effective and intuitive strategy for studying the regulatory mechanism of gene expression., (© 2024 John Wiley & Sons Ltd.)

Achaete-Scute Complex Homolog 1 (ASCL1) is a key regulator in the development and function of the nervous system, particularly in the process of neuronal and neuroendocrine cell differentiation. By employing the CRISPR/Cas9 system, we successfully established an ASCL1-mCherry knock-in human embryonic stem cell (hESC) line by inserting a P2A-mCherry fragment at the ASCL1 locus. The mCherry reporter effectively demonstrated the expression level of endogenous ASCL1 during the process of inducing pulmonary neuroendocrine cells (PNECs) from hESC. This reporter cell line holds significant value as a research tool for investigating the process of lung neuroendocrine cell differentiation, conducting drug screening, and exploring the underlying mechanisms of lung diseases associated with PNECs dysfunction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: Cancer stem cells (CSCs) account for recurrence and resistance to breast cancer drugs, rendering them a cause of mortality and therapeutic failure. In this study, we examined the effects of exposure to low concentrations of doxorubicin (Dox) on CSCs and non-CSCs from TNBC., Methods: The effects of Dox were studied using the SORE6 reporter system. We examined the enrichment of the CSCs population, as well as the proliferation, and death of the reporter-positive fraction (GFP + cells) by flow cytometry. The resistant and stemness phenotypes were analyzed by viability and mammosphere formation assay, respectively. We identified differentially expressed and coregulated genes by RNA-seq analysis, and the correlation between gene expression and clinical outcome was evaluated by Kaplan-Mayer analysis using public databases., Results: In MDAMB231 and Hs578t cells, we identified enriched subsets in the CSCs population after continuous exposure to low concentrations of Dox. Cells from these enriched cultures showed resistance to toxic concentrations of Dox and increased efficiency of mammosphere formation. In purified GFP + or GFP- cells, Dox increased the mammosphere-forming efficiency, promoted phenotypic switches in non-CSCs populations to a CSC-like state, reduced proliferation, and induced differential gene expression. We identified several biological processes and molecular functions that partially explain the development of doxorubicin-resistant cells and cellular plasticity. Among the genes that were regulated by Dox exposure, the expression of ITGB1, SNAI1, NOTCH4, STAT5B, RAPGEF3, LAMA2, and GNAI1 was significantly associated with poor survival, the stemness phenotype, and chemoresistance., Conclusion: The generation of chemoresistant cells that have characteristics of CSCs, after exposure to low concentrations of Dox, involves the differential expression of genes that have a clinical impact., (© 2024. The Author(s).)

We generated a human induced pluripotent stem cell (hiPSC) line (CMCi014-A-78) expressing a GFP reporter in the 3'-UTR region of the KLOTHO locus using CRISPR/Cas9-mediated homologous recombination to screen for candidates regulating KLOTHO. The established cell line exhibits a normal karyotype, typical stem cell morphology, expression of pluripotency markers, and the ability to differentiate into the three germ layers. Consequently, this hiPSC line could serve as a valuable resource for screening KLOTHO regulators in hiPSC-derived target cells or organoids., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The objective of the current study is to develop a new method for tracking transplanted human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) using magnetic resonance imaging (MRI). The CRISPR/dCas9 activation system is employed to overexpress ferritin heavy chain (FHC) in hiPSC-CMs. The mRNA and protein expression of FHC in hiPSC and hiPSC-CMs significantly increased after transfection. Iron chloride does not affect the cell viability in a concentration range from 0 to 2000 µm. hiPSCs overexpressing FHC (hiPSC- FHC OE ) and hiPSC-CMs overexpressing FHC (hiPSC-CM-FHC OE ) significantly enhanced cellular uptake of iron chloride but with no changes in electrophysiological properties compared to hiPSC-CM-Control. Furthermore, hiPSC-CM-FHC OE presented robust contrast and lower T 2 * values, signifying their potential as highly effective candidates for cardiac MRI. Next, hiPSC-CM-FHC OE is injected into mouse hearts and after 3 days of transplantation, MR images are obtained. hiPSC-CM-FHC OE cells exhibited clear signals in the hearts with lower T 2 * and rapid signal decay. Collectively, data from this proof-of-concept study demonstrated that endogenous labeling with FHC in hiPSC-CMs can be a potent strategy for enhancing the accuracy of cardiac MRI. This technology represents a significant step forward in tracking the transplanted hiPSC-CMs in the hearts of live animals., (© 2024 Wiley‐VCH GmbH.)

Cancer stem cells (CSC) play an important role in carcinogenesis and are acknowledged to be responsible for chemoresistance in cholangiocarcinoma (CCA). Studying CCA CSC has been challenging, due to lack of consensus CSC markers, and to their plastic nature. Since dual expression of the core pluripotent factors SOX2/OCT4 has been shown to correlate with poor outcome in CCA patients, we selected the SOX2/OCT4 activating short half-life GFP-based live reporter (SORE6-dsCopGFP) to study CSC dynamics at the single-cell level. Transduction of five human CCA cell lines resulted in the expression of 1.8-13.1% GFP-positive (SORE6 POS ) cells. By live imaging, we found that SORE6 POS CCA cells possess self-renewal capacity and that they can be induced to differentiate. Significantly, the SORE6 POS cells were highly tumorigenic, both in vitro and in vivo, thus implicating the characteristics of primary CSCs. When we then analyzed for selected CSC-related markers, we found that the majority of both CD133 + /CD44 + , and CD133 + /LGR5 + CCA cells were SORE6 POS cells. Exposing transduced cells to standard CCA chemotherapy revealed higher growth rate inhibition at 50% (GR 50 s) for SORE6 POS cells compared to GFP-negative (SORE6 NEG ) ones indicating that these CSC-like cells were more resistant to the treatment. Moreover, the chemotherapy induced SORE6 POS from SORE6 NEG cells, while retaining the existing SORE6 POS population. Finally, treatment of transduced cells with CDK4/6 inhibitors in vitro for 3 days resulted in a lowered CSC number in the culture. Thus, applying a live reporter system allowed us to elucidate the stem cell diversity and drug-induced plasticity of CCA CSCs. These findings have clear implications for future management of such patients., (© 2024. The Author(s).)

Mitochondria-endoplasmic reticulum contact sites (MERCS) serve as hotspots for important cellular processes, including calcium homeostasis, phospholipid homeostasis, mitochondria dynamics, and mitochondrial quality control. MERCS reporters based on complementation of green fluorescent proteins (GFP) fragments have been designed to visualize MERCS in real-time, but we find that they do not accurately respond to changes in MERCS content. Here, we utilize split LacZ complementing fragments to develop the first MERCS reporter system (termed SpLacZ-MERCS) that continuously integrates the MERCS information within a cell and generates a fluorescent output. Our system exhibits good organelle targeting, no artifactual tethering, and effective, dynamic tracking of the MERCS level in single cells. The SpLacZ-MERCS reporter was validated by drug treatments and genetic perturbations known to affect mitochondria-ER contacts. The signal-integrating nature of SpLacZ-MERCS may enable systematic identification of genes and drugs that regulate mitochondria-ER interactions. Our successful application of the split LacZ complementation strategy to study MERCS may be extended to study other forms of interorganellar crosstalk.

Oropouche fever caused by Oropouche virus (OROV) is a significant zoonosis in Central and South America. Despite its public health significance, we lack high-throughput diagnostics, therapeutics, and a comprehensive knowledge of OROV biology. Reporter viruses are valuable tools to rapidly study virus dynamics and develop neutralization and antiviral screening assays. OROV is a tri-segmented bunyavirus, which makes generating a reporter virus challenging, as introducing foreign elements into the viral genome typically affects fitness. We previously demonstrated that the non-structural gene NSm on the OROV medium (M) segment is non-essential for replication in vitro . Taking advantage of this, we have now generated a recombinant OROV expressing fluorescent protein ZsGreen in place of NSm. This reporter OROV is both stable and pathogenic in IFNAR -/- mice and provides a powerful tool for OROV pathogenesis studies and assay development.IMPORTANCEEmerging and reemerging infectious agents such as zoonotic bunyaviruses are of global health concern. Oropouche virus (OROV) causes recurring outbreaks of acute febrile illness in the Central and South American human populations. Biting midges are the primary transmission vectors, whereas sloths and non-human primates are their reservoir hosts. As global temperatures increase, we will likely see an expansion in arthropod-borne pathogens such as OROV. Therefore, developing reagents to study pathogen biology to aid in identifying druggable targets is essential. Here, we demonstrate the feasibility and use of a fluorescent OROV reporter in mice to study viral dynamics and pathogenesis. We show that this reporter OROV maintains characteristics such as growth and pathogenicity similar to the wild-type virus. Using this reporter virus, we can now develop methods to assist OROV studies and establish various high-throughput assays., Competing Interests: The authors declare no conflict of interest.

Here, we introduce 'TICIT', targeted integration by CRISPR-Cas9 and integrase technologies, which utilizes the site-specific DNA recombinase - phiC31 integrase - to insert large DNA fragments into CRISPR-Cas9 target loci. This technique, which relies on first knocking in a 39-basepair phiC31 landing site via CRISPR-Cas9, enables researchers to repeatedly perform site-specific transgenesis at the exact genomic location with high precision and efficiency. We applied this approach to devise a method for the instantaneous determination of a zebrafish's genotype simply by examining its color. When a zebrafish mutant line must be propagated as heterozygotes due to homozygous lethality, employing this method allows facile identification of a population of homozygous mutant embryos even before the mutant phenotypes manifest. Thus, it should facilitate various downstream applications, such as large-scale chemical screens. We demonstrated that TICIT could also create reporter fish driven by an endogenous promoter. Further, we identified a landing site in the tyrosinase gene that could support transgene expression in a broad spectrum of tissue and cell types. In sum, TICIT enables site-specific DNA integration without requiring complex donor DNA construction. It can yield consistent transgene expression, facilitate diverse applications in zebrafish, and may be applicable to cells in culture and other model organisms., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)