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Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies.

Multimodality reporter gene imaging combines the sensitivity, resolution and translational potential of two or more signals. The approach has not been widely adopted by the animal imaging community, mainly because its utility in this area is unproven. We developed a new complementation-based reporter gene system where the large component of split NanoLuc luciferase (LgBiT) presented on the surface of cells (TM-LgBiT) interacts with a radiotracer consisting of the high-affinity complementary HiBiT peptide labeled with a radionuclide. Radiotracer uptake could be imaged in mice using SPECT/CT and bioluminescence within two hours of implanting reporter-gene-expressing cells. Imaging data were validated by ex vivo biodistribution studies. Following the demonstration of complementation between the TM-LgBiT protein and HiBiT radiotracer, we validated the use of the technology in the highly specific in vivo multimodal imaging of cells. These findings highlight the potential of this new approach to facilitate the advancement of cell and gene therapies from bench to clinic. [ABSTRACT FROM AUTHOR]

Objective To investigate the feasibility of granzyme B (GB) promoter-controlled ferritin heavy chain (FTH1) reporter gene expression for monitoring the activation status of chimeric antigen receptor T cells (CAR-T) by magnetic resonance imaging (MRI). Methods Cytotoxic T lymphocytes (CTLs) were screened by Ficoll density gradient centrifugation and flow sorting. The GB promoter and FTH1 gene were ligated together with disialoganglioside 2 (GD2) CAR, and lentiviral vectors were transfected into CTLs to construct GD2-CAR-T/pGB-FTH1 cells. GD2-CAR-T/pCMV-FTH1, GD2-CAR-T, and T cells served as control cells. CytoTox96@non-radioactive cytotoxicity was used to detect the killing effect of each group of cells after co-culture with human neuroblastoma cells (SK-N-SH). ELISA was employed to detect the coincubation factor as well as the amount of GB secretion. Western blotting, Prussian blue staining and cellular MRI were applied to detect the expression of the FTH1 gene after co-culture. Results CTLs were successfully obtained, and then GD2-CAR-T/pGB-FTH1, GD2-CAR-T/pCMV-FTH1 and GD2-CAR-T cells were constructed. The killing effect, co-incubation factor and GB secretion of the above 3 groups of cells were significantly higher than those of the T cells, and the level of GB expression was highest at day 1, and then decreased in order at day 3 and day 7 after co-culturing with SK-N-SH cells. The relative expression of FTH1 and iron content of the GD2-CAR-T/pGB-FTH1 cells showed the same trend as GB expression, and the MRI signals were gradually increased. There were no significant differences in the relative expression of FTH1, iron content and MRI signals in the GD2-CAR-T/pCMV-FTH1 cells at all time points. No FTH1 expression or iron aggregation was observed in the GD2-CAR-T and T cells groups. Conclusion MRI based on the FTH1 reporter gene driven by the granzyme B promoter can reflect the GB expression level and tumor killing effect of CAR-T cells, which provides a potential real-time visual means to monitor the cell activation status for CAR-T therapy.

The ability to regulate the expression of genes is a central tool for the characterization of fungal genes. This is of particular interest to study genes required for specific processes or the effect of genes expressed only under specific conditions. Saccharomycopsis species show a unique property of necrotrophic mycoparasitism that is activated upon starvation. Here we describe the use of the MET17 promoter of S. schoenii as a tool to regulate gene expression based on the availability of methionine. Conditional expression was tested using lacZ and GFP reporter genes. Gene expression could be strongly down-regulated by the addition of methionine or cysteine to the growth medium and upregulated by starvation for methionine. We used X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) to detect lacZ-expression in plate assays and ONPG (ortho-nitrophenyl-β-galactopyranoside) as a substrate for β-galactosidase in liquid-phase assays. For in vivo expression analyses we used fluorescence microscopy for the detection and localization of a MET17-driven histone H4-GFP reporter gene. With these assays we demonstrated the usefulness of the MET17 promoter to regulate expression of genes based on methionine availability. In silico analyses revealed similar promoter motifs as found in MET3 genes of Saccharomyces cerevisiae and Ashbya gossypii. This suggests a regulation of the MET17 promoter by CBF1 and MET31/MET32 in conjunction with the transcriptional activator MET4, which were also identified in the S. schoenii genome. Summary: This article describes the characterization of the S. schoenii MET17 promoter for regulated gene expression. [ABSTRACT FROM AUTHOR]

Key message: A robust agroinfiltration-mediated transient gene expression method for soybean leaves was developed. Plant genotype, developmental stage and leaf age, surfactant, and Agrobacterium culture conditions are important for successful agroinfiltration. Agroinfiltration of Nicotiana benthamiana has emerged as a workhorse transient assay for plant biotechnology and synthetic biology to test the performance of gene constructs in dicot leaves. While effective, it is nonetheless often desirable to assay transgene constructs directly in crop species. To that end, we innovated a substantially robust agroinfiltration method for Glycine max (soybean), the most widely grown dicot crop plant in the world. Several factors were found to be relevant to successful soybean leaf agroinfiltration, including genotype, surfactant, developmental stage, and Agrobacterium strain and culture medium. Our optimized protocol involved a multi-step Agrobacterium culturing process with appropriate expression vectors, Silwet L-77 as the surfactant, selection of fully expanded leaves in the VC or V1 stage of growth, and 5 min of vacuum at − 85 kPa followed by a dark incubation period before plants were returned to normal growth conditions. Using this method, young soybean leaves of two lines—V17-0799DT, and TN16-5004—were high expressors for GUS, two co-expressed fluorescent protein genes, and the RUBY reporter product, betalain. This work not only represents a new research tool for soybean biotechnology, but also indicates critical parameters for guiding agroinfiltration optimization for other crop species. We speculate that leaf developmental stage might be the most critical factor for successful agroinfiltration. [ABSTRACT FROM AUTHOR]

Abstract Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies.

Purpose: Chimeric antigen receptor (CAR) T cell therapy has achieved great success in treating hematologic malignancies. However, it is yet to prove effective in the treatment of solid tumors. Thus, it is necessary to develop appropriate methodology for the long-term, accurate, and quantitative evaluation of the distribution and activities of CAR T cells in solid tumors. In the present study, we engineered TfR ΔPSMA CAR (CAR-ΔPSMA) T cells, which targeted the transferrin receptor (TfR) expressed by tumor cells and could be tracked in vivo via a reporter gene encoding the truncated prostate specific membrane antigen (ΔPSMA). We then quantitatively monitored these CAR T cells in vitro and in vivo using [68Ga]Ga-PSMA-617 positron emission tomography (PET)/computed tomography (CT). Methods: The CAR-ΔPSMA T cells were genetically engineered by transducing T cells with a lentiviral vector encoding TfR41BBζ-T2A-ΔPSMA. Firstly, the target expression, activation, and cytotoxicity of CAR-ΔPSMA T cells were validated in vitro. Secondly, the minimum thresholds of CAR-ΔPSMA T cells detection for [68Ga]Ga-PSMA-617 PET/CT were also determined in vitro and in vivo respectively. Lastly, the feasibility of monitoring the biodistribution and infiltration of CAR-ΔPSMA T cells after systematic administration was evaluated in the breast cancer subcutaneous xenograft model. Results: The CAR-ΔPSMA T cells retained activation and tumor killing capacity after transduction of the ΔPSMA-encoding reporter gene. Next, the CAR-ΔPSMA T cells could be reliably tracked by [68Ga]Ga-PSMA-617 PET/CT, the detection sensitivity of which was 250 cells/mm3 in vitro and 100 cells/mm3 in vivo. Next, the sequential imaging assays revealed that [68Ga]Ga-PSMA-617 PET/CT could be used to specifically visualize ΔPSMA+ CAR T cells at the tumor site. The increase in the [68Ga]Ga-PSMA-617 signal intensity over time allowed us to effectively detect CAR T cells in vivo. Conclusion: Our findings preliminarily confirmed that [68Ga]Ga-PSMA-617 PET/CT could reliably detect CAR-ΔPSMA T cells in vitro and in vivo in solid tumors, laying the foundation for the monitoring CAR T cell therapy in the future. [ABSTRACT FROM AUTHOR]

Glyoxylate shunt is an important pathway for microorganisms to survive under multiple stresses. One of its enzymes, malate synthase (encoded by aceB gene), has been widely speculated for its contribution to both the pathogenesis and virulence of various microorganisms. We have previously demonstrated that malate synthase (MS) is required for the growth of Salmonella Typhimurium (S. Typhimurium) under carbon starvation and survival under oxidative stress conditions. The aceB gene is encoded by the acetate operon in S. Typhimurium. We attempted to study the activity of acetate promoter under both the starvation and oxidative stress conditions in a heterologous system. The lac promoter of the pUC19 plasmid was substituted with the putative promoter sequence of the acetate operon of S. Typhimurium upstream to the lacZ gene and transformed the vector construct into E. coli NEBα cells. The transformed cells were subjected to the stress conditions mentioned above. We observed a fourfold increase in the β-galactosidase activity in these cells resulting from the upregulation of the lacZ gene in the stationary phase of cell growth (nutrient deprived) as compared to the mid-log phase. Following exposure of stationary phase cells to hypochlorite-induced oxidative stress, we further observed a 1.6-fold increase in β galactosidase activity. These data suggest the induction of promoter activity of the acetate operon under carbon starvation and oxidative stress conditions. Thus, these observations corroborate our previous findings regarding the upregulation of aceB expression under stressful environments. [ABSTRACT FROM AUTHOR]

In solid tumors, there are multiple barriers for a chimeric antigen receptor (CAR) T cell to surmount in order to reach the tumor site. For better understanding whether CAR T cells effectively infiltrate into tumor site, and simultaneously, whether there are off-target effects, real-time monitoring technologies need to be established. Cell-based positron emission tomography reporter genes have been developed to monitor engineered cells in living subjects. In this study, we reported the construction of a novel reporter gene truncated prostate-specific membrane antigen (ΔPSMA) pending for monitoring CAR T cells using 68Ga-PSMA-617 and a method for tracking the distribution of CAR T cells in vivo was developed. Data were provided to demonstrate that ΔPSMA was predominantly localized on the plasma membrane and could take up 68Ga-PSMA-617 in vitro in a time-dependent manner. And the expression of ΔPSMA did not affect CAR expression and cytolytic capacity of CAR T cells. CAR-ΔPSMA T cell xenografts in nude mice were clearly imaged by positron emission tomography 60 min after injection of 68Ga-PSMA-617. PSMA paired with 68Ga-PSMA-617 was capable of identifying approximately 1 × 104 engineered CAR T cells. The ability to image small numbers of CAR T cells in vivo would be helpful to accelerate the translation of cell-based therapies into the clinic, and it may reinforce our understanding of treatment success, failure, and toxicity. [ABSTRACT FROM AUTHOR]

Genetic tools form the basis for the study of molecular mechanisms. Despite many recent advances in the field of genetic engineering in bacteria, genetic toolsets remain scarce for non-model organisms, such as the obligatory human pathogen Streptococcus pyogenes. To overcome this limitation and enable the straightforward investigation of gene functions in S. pyogenes, we have developed a comprehensive genetic toolset. By adapting and combining different tools previously applied in other Gram-positive bacteria, we have created new replicative and integrative plasmids for gene expression and genetic manipulation, constitutive and inducible promoters as well as fluorescence reporters for S. pyogenes. The new replicative plasmids feature low- and high-copy replicons combined with different resistance cassettes and a standardized multiple cloning site for rapid cloning procedures. We designed site-specific integrative plasmids and verified their integration by nanopore sequencing. To minimize the effect of plasmid integration on bacterial physiology, we screened publicly available RNA-sequencing datasets for transcriptionally silent sites. We validated this approach by designing the integrative plasmid pSpy0K6 targeting the transcriptionally silent gene SPy_1078. Analysis of the activity of different constitutive promoters indicated a wide variety of strengths, with the lactococcal promoter P23 showing the strongest activity and the synthetic promoter PxylS2 showing the weakest activity. Further, we assessed the functionality of three inducible regulatory elements including a zinc- and an IPTG-inducible promoter as well as an erythromycin-inducible riboswitch that showed low-to-no background expression and high inducibility. Additionally, we demonstrated the applicability of two codon-optimized fluorescent proteins, mNeongreen and mKate2, as reporters in S. pyogenes. We therefore adapted the chemically defined medium called RPMI4Spy that showed reduced autofluorescence and enabled efficient signal detection in plate reader assays and fluorescence microscopy. Finally, we developed a plasmid-based system for genome engineering in S. pyogenes featuring the counterselection marker pheS*, which enabled the scarless deletion of the sagB gene. This new toolbox simplifies previously laborious genetic manipulation procedures and lays the foundation for new methodologies to study gene functions in S. pyogenes, leading to a better understanding of its virulence mechanisms and physiology.

In bacteria, the Rho protein mediates Rho-dependent termination (RDT) by identifying a non-specific cytosine-rich Rho utilization site on the newly synthesized RNA. As a result of RDT, downstream RNA transcription is reduced. Due to the bias in reverse transcription and PCR amplification, we could not identify the RDT site by directly measuring the amount of mRNA upstream and downstream of RDT sites. To overcome this difficulty, we employed a 77 bp reporter gene argX, (coding tRNAarg) from Brevibacterium albidum, and we transcriptionally fused it to the sequences to be assayed. We constructed a series of plasmids by combining a segment of the galactose (gal) operon sequences, both with and without the RDT regions at the ends of cistrons (galE, galT, and galM) upstream of argX. The RNA polymerase will transcribe the gal operon sequence and argX unless it encounters the RDT encoded by the inserted sequence. Since the quantitative real-time PCR (qRT-PCR) method detects the steady state following mRNA synthesis and degradation, we observed that tRNAarg is degraded at the same rate in these transcriptional fusion plasmids. Therefore, the amount of tRNAarg can directly reflect the mRNA synthesis. Using this approach, we were able to effectively assay the RDTs and Rho-independent termination (RIT) in the gal operon by quantifying the relative amount of tRNAarg using qRT-PCR analyses. The resultant RDT% for galET, galTK, and at the end of galM were 36, 26, and 63, individually. The resultant RIT% at the end of the gal operon is 33%. Our findings demonstrate that combining tRNAarg with qRT-PCR can directly measure RIT, RDT, or any other signal that attenuates transcription efficiencies in vivo, making it a useful tool for gene expression research. [ABSTRACT FROM AUTHOR]

ABSTRACTBluetongue virus (BTV) is the causative agent of the important livestock disease bluetongue (BT), which is transmitted via Culicoides bites. BT causes severe economic losses associated with its considerable impact on health and trade of animals. By reverse genetics, we have designed and rescued reporter-expressing recombinant (r)BTV expressing NanoLuc luciferase (NLuc) or Venus fluorescent protein. To generate these viruses, we custom synthesized a modified viral segment 5 encoding NS1 protein with the reporter genes located downstream and linked by the Porcine teschovirus-1 (PTV-1) 2A autoproteolytic cleavage site. Therefore, fluorescent signal or luciferase activity is only detected after virus replication and expression of non-structural proteins. Fluorescence or luminescence signals were detected in cells infected with rBTV/Venus or rBTV/NLuc, respectively. Moreover, the marking of NS2 protein confirmed that reporter genes were only expressed in BTV-infected cells. Growth kinetics of rBTV/NLuc and rBTV/Venus in Vero cells showed replication rates similar to those of wild-type and rBTV. Infectivity studies of these recombinant viruses in IFNAR(−/−) mice showed a higher lethal dose for rBTV/NLuc and rBTV/Venus than for rBTV indicating that viruses expressing the reporter genes are attenuated in vivo. Interestingly, luciferase activity was detected in the plasma of viraemic mice infected with rBTV/NLuc. Furthermore, luciferase activity quantitatively correlated with RNAemia levels of infected mice throughout the infection. In addition, we have investigated the in vivo replication and dissemination of BTV in IFNAR (−/−) mice using BTV/NLuc and non-invasive in vivo imaging systems.IMPORTANCEThe use of replication-competent viruses that encode a traceable fluorescent or luciferase reporter protein has significantly contributed to the in vitro and in vivo study of viral infections and the development of novel therapeutic approaches. In this work, we have generated rBTV that express fluorescent or luminescence proteins to track BTV infection both in vitro and in vivo. Despite the availability of vaccines, BTV and other related orbivirus are still associated with a significant impact on animal health and have important economic consequences worldwide. Our studies may contribute to the advance in orbivirus research and pave the way for the rapid development of new treatments, including vaccines.

Ex vivo programming of T cells can be efficacious but is complex and expensive; therefore, the development of methods to transfect T cells in situ is important. We developed and optimized anti-CD3-targeted lipid nanoparticles (aCD3-LNPs) to deliver tightly packed, reporter gene mRNA specifically to T cells. In vitro, targeted LNPs efficiently delivered mCherry mRNA to Jurkat T cells, and T-cell activation and depletion were associated with aCD3 antibody coating on the surface of LNPs. aCD3-LNPs, but not non-targeted LNPs, accumulated within the spleen following systemic injection, with mCherry and Fluc signals visible within 30 min after injection. At 24 h after aCD3-LNP injection, 2-4% of all splenic T cells and 2-7% of all circulating T cells expressed mCherry, and this was dependent on aCD3 coating density. Targeting and transfection were accompanied by systemic CD25+, OX40+, and CD69+ T-cell activation with temporary CD3e ligand loss and depletion of splenic and circulating subsets. Migration of splenic CD8a+ T cells from the white-pulp to red-pulp, and differentiation from naïve to memory and effector phenotypes, followed upon aCD3-LNP delivery. Additionally, aCD3-LNP injection stimulated the secretion of myeloid-derived chemokines and T-helper cytokines into plasma. Lastly, we administered aCD3-LNPs to tumor bearing mice and found that transfected T cells localized within tumors and tumor-draining lymph nodes following immunotherapy treatment. In summary, we show that CD3-targeted transfection is feasible, yet associated with complex immunological consequences that must be further studied for potential therapeutic applications.

Multimodality reporter gene imaging combines the sensitivity, resolution and translational potential of two or more signals. The approach has not been widely adopted by the animal imaging community, mainly because its utility in this area is unproven. We developed a new complementation-based reporter gene system where the large component of split NanoLuc luciferase (LgBiT) presented on the surface of cells (TM-LgBiT) interacts with a radiotracer consisting of the high-affinity complementary HiBiT peptide labeled with a radionuclide. Radiotracer uptake could be imaged in mice using SPECT/CT and bioluminescence within two hours of implanting reporter-gene-expressing cells. Imaging data were validated by ex vivo biodistribution studies. Following the demonstration of complementation between the TM-LgBiT protein and HiBiT radiotracer, we validated the use of the technology in the highly specific in vivo multimodal imaging of cells. These findings highlight the potential of this new approach to facilitate the advancement of cell and gene therapies from bench to clinic.

CRISPR-Cas-mediated site-specific integration of transgenes by homology-directed repair (HDR) is challenging, especially in primary cells, where inferior editing efficiency may impede the development of gene- and cellular therapies. Various strategies for enrichment of cells with transgene integrations have been developed, but most strategies either generate unwanted genomic scars or rely on permanent integration and expression of a reporter gene used for selection. However, stable expression of a reporter gene may perturb cell homeostasis and function. Here we develop a broadly applicable and versatile enrichment strategy by harnessing the capability of CRISPR activation (CRISPRa) to transiently induce expression of a therapeutically relevant reporter gene used for immunomagnetic enrichment. This strategy is readily adaptable to primary human T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs), where enrichment of 1.8- to 3.3-fold and 3.2- to 3.6-fold was achieved, respectively. Furthermore, chimeric antigen receptor (CAR) T cells were enriched 2.5-fold and demonstrated improved cytotoxicity over non-enriched CAR T cells. Analysis of HDR integrations showed a proportion of cells harboring deletions of the transgene cassette arising either from impartial HDR or truncated adeno-associated virus (AAV) vector genomes. Nonetheless, this novel enrichment strategy expands the possibility to enrich for transgene integrations in research settings and in gene and cellular therapies.

The study of neural circuits, which underlies perception, cognition, emotion, and behavior, is essential for understanding the mammalian brain, a complex organ consisting of billions of neurons. To study the structure and function of the brain, in vivo neuronal labeling and imaging techniques are crucial as they provide true physiological information that ex vivo methods cannot offer. In this paper, we present a new strategy for in vivo neuronal labeling and quantification using MRI. We demonstrate the efficacy of this method by delivering the oatp1a1 gene to the target neurons using rAAV2-retro virus. OATP1A1 protein expression on the neuronal membrane increased the uptake of a specific MRI contrast agent (Gd-EOB-DTPA), leading to hyperintense signals on T1W images of labeled neuronal populations. We also used dynamic contrast enhancement-based methods to obtain quantitative information on labeled neuronal populations in vivo.

Bioreporters are live natural or genetically engineered, bacteria, cyanobacteria, yeast, plant, or mammalian cells that produce signals in response to a specific environment. The generated signals could be measured and used as a sensor to detect and monitor target chemical or physical agents. The recombinant DNA technology is employed to design a bioreporter cell of choice, which mainly involves the fusion of a reporter gene with a promoter gene, activated by some environmental stimulus or set of genes to produce a variety of distinguishable signal that denotes the presence of a chemical or physical agent. The commonly used reporter genes are bioluminescent luciferase (lux or luc), green fluorescent protein, aequorin, and β-galactosidase (lacZ), which is a colorimetric signa l ling element. The bioreporters must remain viable in a range of environmental testing regimes when introduced. They can be deployed for rapid, easy, and economical detection, and in some cases, real-time to nearly real-time measurement potential, thus providing a promising aspect in the detection and removal of environmental pollutants. This review focuses on the principles, mechanisms, and performance of various bioreporters and the advancements made in applications of bioreporter in managing the environment. [ABSTRACT FROM AUTHOR]

Imaging contrast agents are widely investigated in preclinical and clinical studies, among which biogenic imaging contrast agents (BICAs) are developing rapidly and playing an increasingly important role in biomedical research ranging from subcellular level to individual level. The unique properties of BICAs, including expression by cells as reporters and specific genetic modification, facilitate various in vitro and in vivo studies, such as quantification of gene expression, observation of protein interactions, visualization of cellular proliferation, monitoring of metabolism, and detection of dysfunctions. Furthermore, in human body, BICAs are remarkably helpful for disease diagnosis when the dysregulation of these agents occurs and can be detected through imaging techniques. There are various BICAs matched with a set of imaging techniques, including fluorescent proteins for fluorescence imaging, gas vesicles for ultrasound imaging, and ferritin for magnetic resonance imaging. In addition, bimodal and multimodal imaging can be realized through combining the functions of different BICAs, which helps overcome the limitations of monomodal imaging. In this review, the focus is on the properties, mechanisms, applications, and future directions of BICAs. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a type of RNA virus that belongs to the Flaviviridae family. We have reported the construction of a DNA-launched replicon of the Asian-lineage Natal RGN strain and the production of single-round infectious particles (SRIPs) via the combination of prM/E virus-like particles with the replicon. The main objective of the study was to engineer the ZIKV replicon as mammalian expression vectors and evaluate the potential of ZIKV mini-replicon-based SRIPs as delivery vehicles for heterologous gene expression in vitro and in vivo. The mini-replicons contained various genetic elements, including NS4B, an NS5 methyltransferase (MTase) domain, and an NS5 RNA-dependent RNA polymerase (RdRp) domain. Among these mini-replicons, only ZIKV mini-replicons 2 and 3, which contained the full NS5 and NS4B-NS5 genetic elements, respectively, exhibited the expression of reporters (green fluorescent protein (GFP) and cyan fluorescent protein–yellow fluorescent fusion protein (CYP)) and generated self-replicating RNAs. When the mini-replicons were transfected into the cells expressing ZIKV prM/E, this led to the production of ZIKV mini-replicon-based SRIPs. ZIKV mini-replicon 3 SRIPs showed a significantly higher yield titer and a greater abundance of self-replicating replicon RNAs when compared to ZIKV mini-replicon 2 SRIPs. Additionally, there were disparities in the dynamics of CYP expression and cytotoxic effects observed in various infected cell types between ZIKV mini-replicon 2-CYP and 3-CYP SRIPs. In particular, ZIKV mini-replicon 3-CYP SRIPs led to a substantial decrease in the survival rates of infected cells at a MOI of 2. An in vivo gene expression assay indicated that hACE2 expression was detected in the lung and brain tissues of mice following the intravenous administration of ZIKV mini-replicon 3-hACE2 SRIPs. Overall, this study highlights the potential of ZIKV mini-replicon-based SRIPs as promising vehicles for gene delivery applications in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Plants have evolved signaling mechanisms such as the multi-step phosphorelay (MSP) to respond to different internal and external stimuli. MSP responses often result in gene transcription regulation that is modulated through transcription factors such as B-type Arabidopsis response regulator (ARR) proteins. Among these proteins, ARR2 is a key component that is expressed ubiquitously and is involved in many aspects of plant development. Although it has been noted that B-type ARRs bind to their cognate genes through a DNA-binding domain termed the GARP domain, little is known about the structure and function of this type of DNA-binding domain; thus, how ARRs bind to DNA at a structural level is still poorly understood. In order to understand how the MSP functions in planta, it is crucial to unravel both the kinetics as well as the structural identity of the components involved in such interactions. For this reason, this work focusses on resolving how the GARP domain of ARR2 (GARP2) binds to the promoter region of ARR5, one of its native target genes in cytokinin signaling. We have established that GARP2 specifically binds to the ARR5 promoter with three different bi-molecular interaction systems—qDPI-ELISA, FCS, and MST—and we also determined the KD of this interaction. In addition, structural modeling of the GARP2 domain confirms that GARP2 entails a HTH motif, and that protein–DNA interaction most likely occurs via the α3-helix and the N-terminal arm of this domain since mutations in this region hinder ARR2's ability to activate transcription. [ABSTRACT FROM AUTHOR]

Dengue virus, which belongs to the Flaviviridae family, can induce a range of symptoms from mild to severe, including dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. While infectious cloning technology is a useful tool for understanding viral pathogenesis and symptoms, it exhibits limitations when constructing the entire Flavivirus genome. The instability and toxicity of the genome to bacteria make its full‐length construction in bacterial vectors a time‐consuming and laborious process. To address these challenges, we employed the modified infectious subgenomic amplicon (ISA) method in this study, which can potentially be a superior tool for reverse genetic studies on the dengue virus. Using ISA, we generated recombinant dengue viruses de novo and validated their robust replication in both human and insect cell lines, which was comparable to that of the original strains. Moreover, the efficiency of ISA in genetically modifying the dengue virus was elucidated by successfully inserting the gene for green fluorescence protein into the genome of dengue virus serotype 4. Overall, this study highlighted the effectiveness of ISA for genetically engineering the dengue virus and provided a technical basis for a convenient reverse genetics system that could expedite investigations into the dengue virus. [ABSTRACT FROM AUTHOR]

Objective·To establish a T-box transcription factor Brachyury (T gene) fluorescence reporter cell line, in which foot-and-mouth disease virus 2A (F2A) and enhanced green fluorescent protein (EGFP) were knocked in at the end of mouse T gene (mT-F2A-EGFP) in mouse embryonic stem cells (mESCs) by CRISPR/Cas9n (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 nickase)-mediated homologous-directed repair (HDR) technology.Methods·First of all, the specific single guide RNA (sgRNA) plasmid targeting the sequence near the stop codon of the mouse T gene and the plasmid donor containing F2A-EGFP were constructed. These two plasmids were co-delivered into mESCs E14Tg2a (E14) by electroporation. In this way, the desired fluorescent marker EGFP with self-cleaving peptide F2A were introduced into the end of T gene via HDR. Then, the monoclonal cells, obtained after drug selection and verified by sequencing, were induced for differentiation as embryonic bodies (EB), of which the fluorescence signals of mT-F2A-EGFP were monitored by fluorescence microscope and flow cytometry. These reporter clones were also selected before and after differentiation by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), which detected the transcription levels of marker genes determing pluripotency, mesendoderm differentiation or ectoderm differentiation. In addition, the cell cycle and growth curve of these clones were detected. Meanwhile, alkaline phosphatase (AP) staining was used to detect the stem cell characteristics of these candidate clones. Finally, the clone T1 carrying mT-F2A-EGFP was selected for EB differentiation. Flow cytometry was used to sort out EGFP expression cells (EGFP＋) and non-EGFP expressing cells (EGFP－) from the EBs comprising multiple lineage cells upon differentiation, of which cell lineage markers were checked by RT-qPCR.Results·EGFP was correctly inserted after the T gene in E14, whose fluorescence intensity reflected the expression level of endogenous T without observed side effects. When the fluorescence reporter clone T1 was differentiated, the EGFP+ cells sorted by flow cytometry mainly expressed mesendoderm marker genes.Conclusion·The establishment of mESC line carrying mT-F2A-EGFP can realize rapid monitoring of the degree of T regulation, and track mesendoderm cells expressing T marker, EGFP in real time during differentiation.

Abstract Imaging contrast agents are widely investigated in preclinical and clinical studies, among which biogenic imaging contrast agents (BICAs) are developing rapidly and playing an increasingly important role in biomedical research ranging from subcellular level to individual level. The unique properties of BICAs, including expression by cells as reporters and specific genetic modification, facilitate various in vitro and in vivo studies, such as quantification of gene expression, observation of protein interactions, visualization of cellular proliferation, monitoring of metabolism, and detection of dysfunctions. Furthermore, in human body, BICAs are remarkably helpful for disease diagnosis when the dysregulation of these agents occurs and can be detected through imaging techniques. There are various BICAs matched with a set of imaging techniques, including fluorescent proteins for fluorescence imaging, gas vesicles for ultrasound imaging, and ferritin for magnetic resonance imaging. In addition, bimodal and multimodal imaging can be realized through combining the functions of different BICAs, which helps overcome the limitations of monomodal imaging. In this review, the focus is on the properties, mechanisms, applications, and future directions of BICAs.

Abstract Background Transcriptional enhancers are essential for gene regulation, but how these regulatory elements are best defined remains a significant unresolved question. Traditional definitions rely on activity-based criteria such as reporter gene assays, while more recently, biochemical assays based on chromatin-level phenomena such as chromatin accessibility, histone modifications, and localized RNA transcription have gained prominence. Results We examine here whether these two types of definitions, activity-based and chromatin-based, effectively identify the same sets of sequences. We find that, concerningly, the overlap between the two groups is strikingly limited. Few of the data sets we compared displayed statistically significant overlap, and even for those, the degree of overlap was typically small (below 40% of sequences). Moreover, a substantial batch effect was observed in which experiment set rather than experimental method was a primary driver of whether or not chromatin-defined enhancers showed a strong overlap with reporter gene-defined enhancers. Conclusions Our results raise important questions as to the appropriateness of both old and new enhancer definitions, and suggest that new approaches are required to reconcile the poor agreement among existing methods for defining enhancers.

Abstract Thermus thermophilus is an attractive species in the bioindustry due to its valuable natural products, abundant thermophilic enzymes, and promising fermentation capacities. However, efficient and versatile genome editing tools are not available for this species. In this study, we developed an efficient genome editing tool for T. thermophilus HB27 based on its endogenous type I‐B, I‐C, and III‐A/B CRISPR‐Cas systems. First, we systematically characterized the DNA interference capabilities of the different types of the native CRISPR‐Cas systems in T. thermophilus HB27. We found that genomic manipulations such as gene deletion, mutation, and in situ tagging could be easily implemented by a series of genome‐editing plasmids carrying an artificial self‐targeting mini‐CRISPR and a donor DNA responsible for the recombinant recovery. We also compared the genome editing efficiency of different CRISPR‐Cas systems and the editing plasmids with donor DNAs of different lengths. Additionally, we developed a reporter gene system for T. thermophilus based on a heat‐stable β‐galactosidase gene TTP0042, and constructed an engineered strain with a high production capacity of superoxide dismutases by genome modification.

Objective·To establish a T-box transcription factor Brachyury (T gene) fluorescence reporter cell line, in which footand-mouth disease virus 2A (F2A) and enhanced green fluorescent protein (EGFP) were knocked in at the end of mouse T gene (mTF2A-EGFP) in mouse embryonic stem cells (mESCs) by CRISPR/Cas9n (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 nickase)-mediated homologous-directed repair (HDR) technology. Methods·First of all, the specific single guide RNA (sgRNA) plasmid targeting the sequence near the stop codon of the mouse T gene and the plasmid donor containing F2A-EGFP were constructed. These two plasmids were co-delivered into mESCs E14Tg2a (E14) by electroporation. In this way, the desired fluorescent marker EGFP with self-cleaving peptide F2A were introduced into the end of T gene via HDR. Then, the monoclonal cells, obtained after drug selection and verified by sequencing, were induced for differentiation as embryonic bodies (EB), of which the fluorescence signals of mT-F2A-EGFP were monitored by fluorescence microscope and flow cytometry. These reporter clones were also selected before and after differentiation by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), which detected the transcription levels of marker genes determing pluripotency, mesendoderm differentiation or ectoderm differentiation. In addition, the cell cycle and growth curve of these clones were detected. Meanwhile, alkaline phosphatase (AP) staining was used to detect the stem cell characteristics of these candidate clones. Finally, the clone T1 carrying mT-F2A-EGFP was selected for EB differentiation. Flow cytometry was used to sort out EGFP expression cells (EGFP＋) and non-EGFP expressing cells (EGFP－) from the EBs comprising multiple lineage cells upon differentiation, of which cell lineage markers were checked by RT-qPCR. Results·EGFP was correctly inserted after the T gene in E14, whose fluorescence intensity reflected the expression level of endogenous T without observed side effects. When the fluorescence reporter clone T1 was differentiated, the EGFP+ cells sorted by flow cytometry mainly expressed mesendoderm marker genes. Conclusion·The establishment of mESC line carrying mT-F2A-EGFP can realize rapid monitoring of the degree of T regulation, and track mesendoderm cells expressing T marker, EGFP in real time during differentiation. [ABSTRACT FROM AUTHOR]

In vitro experiments have shown that the E2 protein of human papillomaviruses (HPV) binds to the upstream regulatory region (URR) of the viral genome and modulates transcription. Additionally, it seems to be a necessary component for viral DNA replication together with E1. We have developed a transgenic mouse model containing the URR region of the low-risk virus HPV11 that regulates the expression of the lacZ reporter gene. Most interestingly, in these mice, the transgene was exclusively expressed in the bulge region of the hair follicle but not in any other tissues. Further experimental data indicate that in double transgenic mice that also express the HPV11-E2 protein under the control of the Ubiquitin C-promoter, the transcription of the reporter gene is modulated. When E2 is present, the expression of the reporter gene also occurs exclusively in the bulge region of the hair follicles as it does in the single transgenic mice, but the expression of the lacZ driven by the URR is increased and the statistical spread is greater. Even if the expression of the reporter gene occurs in the hair follicles of the dorsal skin of an animal uniform, E2 obviously has the capacity for both to induce and to repress the URR activity in vivo. [ABSTRACT FROM AUTHOR]

Simple Summary: The GL261 murine preclinical model is one of the most commonly used models to study the impact of new GBM treatments. To assess tumor growth, the use of noninvasive imaging is needed. Bioluminescence imaging (BLI) is particularly popular because of its easy access compared to magnetic resonance imaging (MRI). In our study, by comparing in vivo tumor growth of wild-type and luciferase-expressing GL261 models, we observed a difference in the estimation of tumor growth by BLI and MRI in a specific model. The divergence in results was explained by the instability in luciferase expression after transduction. Our study showed that the use of multi-modality imaging prevents possible errors in tumor growth evaluation. Orthotopic glioblastoma xenografts are paramount for evaluating the effect of innovative anti-cancer treatments. In longitudinal studies, tumor growth (or regression) of glioblastoma can only be monitored by noninvasive imaging. For this purpose, bioluminescence imaging (BLI) has gained popularity because of its low cost and easy access. In the context of the development of new nanomedicines for treating glioblastoma, we were using luciferase-expressing GL261 cell lines. Incidentally, using BLI in a specific GL261 glioblastoma model with cells expressing both luciferase and the green fluorescent protein (GL261-luc-GFP), we observed an apparent spontaneous regression. By contrast, the magnetic resonance imaging (MRI) analysis revealed that the tumors were actually growing over time. For other models (GL261 expressing only luciferase and U87 expressing both luciferase and GFP), data from BLI and MRI correlated well. We found that the divergence in results coming from different imaging modalities was not due to the tumor localization nor the penetration depth of light but was rather linked to the instability in luciferase expression in the viral construct used for the GL261-luc-GFP model. In conclusion, the use of multi-modality imaging prevents possible errors in tumor growth evaluation, and checking the stability of luciferase expression is mandatory when using BLI as the sole imaging modality. [ABSTRACT FROM AUTHOR]

Border disease virus (BDV) is a serious pathogen of sheep and goats worldwide. The virus causes considerable economic losses in animal husbandry. Therefore, developing effective and genetic-based therapies to control viral infections, such as BDV, has been remarkable over recent years. A common way to evaluate such therapeutic strategies is by cloning the desired fragment into appropriate vectors to develop cell lines expressing it. Due to the required duties of the NS3 gene for BDV proliferation, this gene's HELICc and nucleotide binding site domain was synthesized and cloned in a lentiviral vector upstream of the GFP gene. The cloning accuracy was verified by digestion with restriction enzymes and sequencing. Thus, the BDV-NS3 HELICc and nucleotide binding site carrying plasmid was prepared successfully and will be applied in a second or third lentiviral packaging system for stable expression of the desired gene. [ABSTRACT FROM AUTHOR]

Cyanobacteria produce a wealth of secondary metabolites. Since these organisms attach fatty acids into molecules in unprecedented ways, cyanobacteria can serve as a novel source for bioactive compounds acting as ligands for Peroxisome Proliferator-Activated Receptors (PPAR). PPARs (PPARα, PPARβ/δ and PPARγ) are ligand-activated nuclear receptors, involved in the regulation of various metabolic and cellular processes, thus serving as potential drug targets for a variety of pathologies. Yet, given that PPARs' agonists can have pan-, dual- or isoform-specific action, some controversy has been raised over currently approved drugs and their side effects, highlighting the need for novel molecules. Here, we expand and validate a cell-based PPAR transactivation activity biosensor, and test it in a screening campaign to guide drug discovery. Biosensor upgrades included the use of different reporter genes to increase signal intensity and stability, a different promoter to modulate reporter gene expression, and multiplexing to improve efficiency. Sensor's limit of detection (LOD) ranged from 0.36–0.89 nM in uniplex and 0.89–1.35 nM in multiplex mode. In triplex mode, the sensor's feature screening, a total of 848 fractions of 96 cyanobacteria extracts were screened. Hits were confirmed in multiplex mode and in uniplex mode, yielding one strain detected to have action on PPARα and three strains to have dual action on PPARα and -β. [ABSTRACT FROM AUTHOR]

Reporter genes are important tools in many biological disciplines. The discovery of novel reporter genes is relatively rare. However, known reporter genes are constantly applied to novel applications. This study reports the performance of the bilirubin-dependent fluorescent protein UnaG from the Japanese eel Anguilla japonicas in live Escherichia coli cells in response to the disruption of outer membrane (OM) integrity at low bilirubin (BR) concentrations. Using the E. coli wild-type strain MC4100, its isogenic OM-deficient mutant strain NR698, and different OM-active compounds, we show that BR uptake and UnaG fluorescence depend on a leaky OM at concentrations of 10 µM BR and below, while fluorescence is mostly OM integrity-independent at concentrations above 50 µM BR. We suggest that these properties of the UnaG–BR couple might be applied as a biosensor as an alternative to the OM integrity assays currently in use. [ABSTRACT FROM AUTHOR]

Cell tracking by in vivo magnetic resonance imaging (MRI) offers a collection of multiple advantages over other imaging modalities, including high spatial resolution, unlimited depth penetration, 3D visualization, lack of ionizing radiation, and the potential for long-term cell monitoring. Three decades of innovation in both contrast agent chemistry and imaging physics have built an expansive array of probes and methods to track cells non-invasively across a diverse range of applications. In this review, we describe both established and emerging MRI cell tracking approaches and the variety of mechanisms available for contrast generation. Emphasis is given to the advantages, practical limitations, and persistent challenges of each approach, incorporating quantitative comparisons where possible. Toward the end of this review, we take a deeper dive into three key application areas – tracking cancer metastasis, immunotherapy for cancer, and stem cell regeneration – and discuss the cell tracking techniques most suitable to each.

In bacteria, the Rho protein mediates Rho-dependent termination (RDT) by identifying a non-specific cytosine-rich Rho utilization site on the newly synthesized RNA. As a result of RDT, downstream RNA transcription is reduced. Due to the bias in reverse transcription and PCR amplification, we could not identify the RDT site by directly measuring the amount of mRNA upstream and downstream of RDT sites. To overcome this difficulty, we employed a 77 bp reporter gene argX, (coding tRNAarg) from Brevibacterium albidum, and we transcriptionally fused it to the sequences to be assayed. We constructed a series of plasmids by combining a segment of the galactose (gal) operon sequences, both with and without the RDT regions at the ends of cistrons (galE, galT, and galM) upstream of argX. The RNA polymerase will transcribe the gal operon sequence and argX unless it encounters the RDT encoded by the inserted sequence. Since the quantitative real-time PCR (qRT-PCR) method detects the steady state following mRNA synthesis and degradation, we observed that tRNAarg is degraded at the same rate in these transcriptional fusion plasmids. Therefore, the amount of tRNAarg can directly reflect the mRNA synthesis. Using this approach, we were able to effectively assay the RDTs and Rho-independent termination (RIT) in the gal operon by quantifying the relative amount of tRNAarg using qRT-PCR analyses. The resultant RDT% for galET, galTK, and at the end of galM were 36, 26, and 63, individually. The resultant RIT% at the end of the gal operon is 33%. Our findings demonstrate that combining tRNAarg with qRT-PCR can directly measure RIT, RDT, or any other signal that attenuates transcription efficiencies in vivo, making it a useful tool for gene expression research.

Many strains of the spirochete Leptospira interrogans serovar Pomona express the osmotically inducible sphingomyelinase gene sph2 at much higher levels than strains from other serovars. We developed a new green fluorescent protein (GFP) reporter plasmid to examine sph2 gene expression determinants. The vector enables the fusion of the test promoter to the ribosome-binding site and coding region of gfp We fused the sph2 promoters from the L. interrogans serovar Lai strain 56601 and from the L. interrogans serovar Pomona strain LC82-25 to gfp to examine the molecular determinants of differential sph2 expression between the two strains. Similar to what was observed with the native sph2 genes, the introduction of the plasmids into the Lai 56601 strain resulted in near background levels of gfp expression from the Lai sph2 promoter, while the expression from the Pomona sph2 promoter was high. The expression of both fusions increased at physiologic levels of osmolarity achieved by adding sodium chloride to the culture medium. We examined the role of a 17-bp upstream element found in all L. interrogans strains expressing low basal levels of sph2 and missing from Pomona strains that express sph2 at high levels. When the 17-bp sequence present upstream of the Lai sph2 promoter was deleted or scrambled, the fusion expression increased substantially. Conversely, the insertion of the 17-bp sequence upstream of the Pomona sph2 promoter diminished fusion expression. In contrast, the removal of an insertion sequence-like element that is found only in the Pomona sph2 upstream sequence had no effect on the expression from the Pomona sph2 fusion in the Lai strain. These findings demonstrate the utility of the gfp reporter plasmid in analyzing gene expression in L. interrogansIMPORTANCE Genetic tools are needed to examine gene expression in the pathogen Leptospira interrogans We developed a reporter plasmid that replicates in L. interrogans with green fluorescent protein (GFP) as the readout of promoter activity. We demonstrated an application of the new reporter plasmid by identifying an upstream element responsible for the poor basal expression of the sph2 sphingomyelinase gene in an L. interrogans serovar Lai strain. This new tool is useful for the discovery of the molecular determinants of L. interrogans gene expression.

Inteins (intervening proteins) are polypeptides that interrupt the sequence of other proteins and remove themselves through protein splicing. In this intein-catalyzed reaction, the two peptide bonds surrounding the intein are rearranged to release the intein from the flanking protein sequences, termed N- and C-exteins, which are concurrently joined by a peptide bond. Because of this unique functionality, inteins have proven exceptionally useful in protein engineering. Previous work has demonstrated that heterologous proteins can be inserted within an intein, with both the intein and inserted protein retaining function, allowing for intein-containing genes to coexpress additional coding sequences. Here, we show that a fluorescent protein (ZsGreen) can be inserted within the Pyrococcus horikoshii RadA intein, with the hybrid protein (ZsG-Int) maintaining fluorescence and splicing capability. We used this system to create a recombinant Ebola virus expressing a fluorescent protein. We first tested multiple potential insertion sites for ZsG-Int within individual Ebola virus proteins, identifying a site within the VP30 gene that facilitated efficient intein splicing in mammalian cells while also preserving VP30 function. Next, we successfully rescued a virus containing the ZsG-Int-VP30 fusion protein, which displayed fluorescence in the infected cells. We thus report a new intein-based application for adding reporters to systems without the need to add additional genes. Further, this work highlights a novel reporter design, whereby the reporter is only made if the protein of interest is translated and does not remain fused to the protein of interest. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a type of RNA virus that belongs to the Flaviviridae family. We have reported the construction of a DNA-launched replicon of the Asian-lineage Natal RGN strain and the production of single-round infectious particles (SRIPs) via the combination of prM/E virus-like particles with the replicon. The main objective of the study was to engineer the ZIKV replicon as mammalian expression vectors and evaluate the potential of ZIKV mini-replicon-based SRIPs as delivery vehicles for heterologous gene expression in vitro and in vivo. The mini-replicons contained various genetic elements, including NS4B, an NS5 methyltransferase (MTase) domain, and an NS5 RNA-dependent RNA polymerase (RdRp) domain. Among these mini-replicons, only ZIKV mini-replicons 2 and 3, which contained the full NS5 and NS4B-NS5 genetic elements, respectively, exhibited the expression of reporters (green fluorescent protein (GFP) and cyan fluorescent protein–yellow fluorescent fusion protein (CYP)) and generated self-replicating RNAs. When the mini-replicons were transfected into the cells expressing ZIKV prM/E, this led to the production of ZIKV mini-replicon-based SRIPs. ZIKV mini-replicon 3 SRIPs showed a significantly higher yield titer and a greater abundance of self-replicating replicon RNAs when compared to ZIKV mini-replicon 2 SRIPs. Additionally, there were disparities in the dynamics of CYP expression and cytotoxic effects observed in various infected cell types between ZIKV mini-replicon 2-CYP and 3-CYP SRIPs. In particular, ZIKV mini-replicon 3-CYP SRIPs led to a substantial decrease in the survival rates of infected cells at a MOI of 2. An in vivo gene expression assay indicated that hACE2 expression was detected in the lung and brain tissues of mice following the intravenous administration of ZIKV mini-replicon 3-hACE2 SRIPs. Overall, this study highlights the potential of ZIKV mini-replicon-based SRIPs as promising vehicles for gene delivery applications in vitro and in vivo.

A strawberry RIPK1, a leu-rich repeat receptor-like protein kinase, is previously demonstrated to be involved in fruit ripening as a positive regulator; however, its role in vegetable growth remains unknown. Here, based on our first establishment of Agrobacterium-mediated transformation of germinating seeds in diploid strawberry by FvCHLH/FvABAR, a reporter gene that functioned in chlorophyll biosynthesis, we got FvRIPK1-RNAi mutants. Downregulation of FvRIPK1 inhibited plant morphogenesis, showing curled leaves; also, this silencing significantly reduced FvABAR and FvABI1 transcripts and promoted FvABI4, FvSnRK2.2, and FvSnRK2.6 transcripts. Interestingly, the downregulation of the FvCHLH/ABAR expression could not affect FvRIPK1 transcripts but remarkably reduced FvABI1 transcripts and promoted FvABI4, FvSnRK2.2, and FvSnRK2.6 transcripts in the contrast of the non-transgenic plants to the FvCHLH/FvABAR-RNAi plants, in which chlorophyll contents were not affected but had abscisic acid (ABA) response in stomata movement and drought stress. The distinct expression level of FvABI1 and FvABI4, together with the similar expression level of FvSnRK2.2 and FvSnRK2.6 in the FvRIPK1- and FvABAR/CHLH-RNAi plants, suggested that FvRIPK1 regulated plant morphogenesis probably by ABA signaling. In addition, FvRIPK1 interacted with FvSnRK2.6 and phosphorylated each other, thus forming the FvRIPK1–FvSnRK2.6 complex. In conclusion, our results provide new insights into the molecular mechanism of FvRIPK1 in plant growth. [ABSTRACT FROM AUTHOR]

Simple Summary: In this study, we generate an innovative endogenous progesterone receptor (PR) reporter gene containing endometrial cancer cell lines and use this tool to visualize PR expression in real-time. We also demonstrated two strategies of using this reporter gene: (1). Drug discovery—screening small molecule inducers for PR expression; (2). Mechanistic investigation—screening potential PR repressors using genome-wide CRISPR knockout library. Expression of progesterone receptor (PR) is a favorable prognostic marker for multiple solid tumors. However, PR expression is reduced or lost in malignant tumors. Thus, monitoring and restoring functional PR expression is important in order to sensitize tumor cells to progesterone therapy in endometrial cancer. We developed stable PR reporter gene containing endometrial cancer cell lines monitoring the endogenous PR expression by inserting mCherry and hygromycin resistant gene at the endogenous PR gene locus by CRISPR/Cas9-mediated genome editing technique. This allows efficient, real-time monitoring of PR expression in its native epigenetic landscape. Reporter gene expression faithfully reflects and amplifies PR expression following treatment with drugs known to induce PR expression. Small molecular PR inducers have been identified from the FDA-approved 1018 drug library and tested for their ability to restore PR expression. Additionally, several candidate PR repressors have been identified by screening the genome-wide CRISPR knockout (GeCKO) library. This novel endogenous PR reporter gene system facilitates the discovery of a new treatment strategy to enhance PR expression and further sensitize progestin therapy in endometrial cancer. These tools provide a systematic, unbiased approach for monitoring target gene expression, allowing for novel drug discovery and mechanistic exploration. [ABSTRACT FROM AUTHOR]

The use of molecular imaging technologies for brain imaging can not only play an important supporting role in disease diagnosis and treatment but can also be used to deeply study brain functions. Recently, with the support of reporter gene technology, optical imaging has achieved a breakthrough in brain function studies at the molecular level. Reporter gene technology based on traditional clinical imaging modalities is also expanding. By benefiting from the deeper imaging depths and wider imaging ranges now possible, these methods have led to breakthroughs in preclinical and clinical research. This article focuses on the applications of magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET) reporter gene technologies for use in brain imaging. The tracking of cell therapies and gene therapies is the most successful and widely used application of these techniques. Meanwhile, breakthroughs have been achieved in the research and development of reporter genes and their imaging probe pairs with respect to brain function research. This paper introduces the imaging principles and classifications of the reporter gene technologies of these imaging modalities, lists the relevant brain imaging applications, reviews their characteristics, and discusses the opportunities and challenges faced by clinical imaging modalities based on reporter gene technology. The conclusion is provided in the last section. [ABSTRACT FROM AUTHOR]

Fluorescent proteins are essential reporters in cell and molecular biology. Here, we found that red-fluorescent proteins possess an alternative translation initiation site that produces a short functional protein isoform in both prokaryotes and eukaryotes. The short isoform creates significant background fluorescence that biases the outcome of expression studies. In this study, we identified the short protein isoform, traced its origin, and determined the extent of the issue within the family of red fluorescent protein. Our analysis showed that the short isoform defect of the red fluorescent protein family may affect the interpretation of many published studies. We provided a re-engineered mCherry variant that lacks background expression as an improved tool for imaging and protein expression studies.

The filamentous fungus Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) is currently being intensively studied as a promising industrial producer of a number of secreted cellulolytic enzymes. In this study, the T. cellulolyticus gene lacA, which encodes a protein orthologous to the fungal extracellular β-galactosidases of family 35, was identified. The substitution of the lacA upstream region with a constitutive promoter demonstrated that the product of this gene is effectively secreted and possesses β-galactosidase activity. The optimal pH and temperature values for the hydrolysis of o-nitrophenyl-β-D-galactopyranoside by this enzyme were determined to be pH 4.5–5.5 and 50 °C, respectively. The negligible production of β-galactosidase activity by strains expressing lacA under native regulation raises the possibility of using lacA as a reporter gene. To test this hypothesis, the native promoter of lacA was replaced with the strong inducible promoter of the T. cellulolyticus cellobiohydrolase I gene. The cultivation of the resulting strain in various media showed that the β-galactosidase activity depends on cultivation conditions similar to the cellobiohydrolase activity. Thus, the suitability of lacA as a reporter for evaluating promoters with a wide range of expression profiles was demonstrated. [ABSTRACT FROM AUTHOR]

The development of imaging techniques in the experimental oncology has made it possible to model and continuously monitor growth and development of tumors in vivo. For this purpose, fluorescent or bioluminescent proteins are most commonly introduced into the genome of target cells which in turn can be used to create xenograft tumor models. In our study, SH-SY5Y human neuroblastoma cells were genetically modified with a firefly luciferase ffLuc gene or far-red fluorescent protein Katushka2S gene, and bioluminescence and fluorescence of obtained cells were analyzed in vitro. Then, produced cell lines were used to create xenograft tumor models in vivo. The bioluminescence and fluorescence of the obtained tumor models were evaluated in vivo. When evaluated in vitro, ffLuc bioluminescence was firstly detected within fewer number of SH-SY5Y cells compared to Katushka2S fluorescence. However, after in vivo administration, the fluorescence intensity of SH-SY5Y-Katushka2S cells was significantly higher than the bioluminescence intensity of the same number of SH-SY5Y-ffLuc cells. This may be due to the distribution and limited intracellular availability of a luciferase substrate, which is administered intraperitoneally, and requires further analysis. [ABSTRACT FROM AUTHOR]

Bioluminescence reporter gene imaging is a robust, high-throughput imaging modality that is useful for tracking cells and monitoring biological processes, both in cell culture and in small animals. We introduced and characterized a novel bioluminescence reporter—membrane-anchored Cypridina luciferase (maCLuc)—paired with a unique vargulin substrate. This luciferase-substrate pair has no cross-reactivity with established d-luciferin- or coelenterazine-based luciferase reporters. We compare maCLuc with several established luciferase-based reporter systems (firefly, click beetle, Renilla, and Gaussia luciferases), using both in vitro and in vivo models. We demonstrate the different imaging characteristics of these reporter systems, which allow for multiplexed-luciferase imaging of 3 and 4 separate targets concurrently in the same animal within 24 h. The imaging paradigms described here can be directly applied for simultaneous in vivo monitoring of multiple cell populations, the activity of selected signal transduction pathways, or a combination of both constitutive and inducible reporter imaging.