Automated In Vivo Phenotypic Screening Platform for Identifying Factors that Affect Cell Regeneration Kinetics.

Various strategies for replacing retinal neurons lost in degenerative diseases are under investigation, including stimulating the endogenous regenerative capacity of Müller Glia (MG) as injury-inducible retinal stem cells. Inherently regenerative species, such as zebrafish, have provided key insights into mechanisms regulating MG dedifferentiation to a stem-like state and the proliferation of MG and MG-derived progenitor cells (MGPCs). Interestingly, promoting MG/MGPC proliferation is not sufficient for regeneration, yet mechanistic studies are often focused on this measure. To fully account for the regenerative process, and facilitate screens for factors regulating cell regeneration, an assay for quantifying cell replacement is required. Accordingly, we adapted an automated reporter-assisted phenotypic screening platform to quantify the pace of cellular regeneration kinetics following selective cell ablation in larval zebrafish. Here, we detail a method for using this approach to identify chemicals and genes that control the rate of retinal cell regeneration following selective retinal cell ablation.

The Role of Microsurgery and Fluorescent-reporter Genes in Establishing Mouse Models for Real-Time Imaging of Metastatic Cancer-Cell Trafficking and Colony Formation: A Revolutionary and Disruptive Technology for Metastasis Research.

The field of experimental microsurgery was pioneered by the great microsurgeon Sun Lee, who developed the foundation of transplant surgery in the clinic. Dr Lee also played a seminal role in introducing microsurgery to establish mouse models of cancer. In 1990, at the age of 70, Dr Lee demonstrated microsurgery techniques to the mouse-model team at AntiCancer Inc., leading to the development of the surgical orthotopic implant (SOI) technique and the first orthotopic mouse models of cancer that metastasized in a pattern similar to clinical cancer. At the beginning of the present century, one of us (NY) from Kanazawa University School of Medicine became a visiting scientist at AntiCancer to learn SOI and develop mouse models of cancer using cancer cells expressing fluorescent reporter genes, such as green fluorescent protein (GFP) and red fluorescent protein (RFP), in order to image metastatic cancer cells trafficking in real time. Since then, a total of eight young surgeons from Kanazawa University have been visiting researchers at AntiCancer, developing SOI mouse models of cancer to visualize cancer cells in vivo, tracking all stages of metastasis in real time. The present perspective review summarizes this seminal work, which has revolutionized the field of metastasis research.

Generation and application of novel hES cell reporter lines for the differentiation and maturation of hPS cell-derived islet-like clusters.

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells, including functional ß-cells, have been based on a detailed understanding of the underlying developmental mechanisms. However, the final differentiation steps, leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells, remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells), which include a lack of ß-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS+ and GCG+ cells and their resolution into mono-hormonal cells (INSeGFP, INSeGFP/GCGmCHERRY) as well as ß-cell maturation (INSeGFP/MAFAmCHERRY) and function (INSGCaMP6). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive, insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions, we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and, using this approach, we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal ß-cells without affecting the number of α-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets.

Cell-Free Translation Quantification via a Fluorescent Minihelix.

Cell-free gene expression systems are used in numerous applications, including medicine making, diagnostics, and educational kits. Accurate quantification of nonfluorescent proteins in these systems remains a challenge. To address this challenge, we report the adaptation and use of an optimized tetra-cysteine minihelix both as a fusion protein and as a standalone reporter with the FlAsH dye. The fluorescent reporter helix is short enough to be encoded on a primer pair to tag any protein of interest via PCR. Both the tagged protein and the standalone reporter can be detected quantitatively in real time or at the end of cell-free expression reactions with standard 96/384-well plate readers, an RT-qPCR system, or gel electrophoresis without the need for staining. The fluorescent signal is stable and correlates linearly with the protein concentration, enabling product quantification. We modified the reporter to study cell-free expression dynamics and engineered ribosome activity. We anticipate that the fluorescent minihelix reporter will facilitate efforts in engineering in vitro transcription and translation systems.

An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal epithelial cells.

The intestinal epithelium dynamically controls cell cycle, yet no experimental platform exists for directly analyzing cell cycle phases in non-immortalized human intestinal epithelial cells (IECs). Here, we present two reporters and a complete platform for analyzing cell cycle phases in live primary human IECs. We interrogate the transcriptional identity of IECs grown on soft collagen, develop two fluorescent cell cycle reporter IEC lines, design and 3D print a collagen press to make chamber slides for optimal imaging while supporting primary human IEC growth, live image cell cycle dynamics, then assemble a computational pipeline building upon free-to-use programs for semi-automated analysis of cell cycle phases. The PIP-FUCCI construct allows for assigning cell cycle phase from a single image of living cells, and our PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths using our publicly available computational pipeline. Treating PIP-FUCCI IECs with oligomycin demonstrates that inhibiting mitochondrial respiration lengthens G1 phase, and PIP-H2A cells allow us to measure that oligomycin differentially lengthens S and G2/M phases across heterogeneous IECs. These platforms provide opportunities for future studies on pharmaceutical effects on the intestinal epithelium, cell cycle regulation, and more.

Context-dependent antisense transcription from a neighboring gene interferes with the expression of mNeonGreen as a functional in vivo fluorescent reporter in the chloroplast of Chlamydomonas reinhardtii.

Advancing chloroplast genetic engineering in Chlamydomonas reinhardtii remains challenging, decades after its first successful transformation. This study introduces the development of a chloroplast-optimized mNeonGreen fluorescent reporter, enabling in vivo observation through a sixfold increase in fluorescence via context-aware construct engineering. Our research highlights the influence of transcriptional readthrough and antisense mRNA pairing on post-transcriptional regulation, pointing to novel strategies for optimizing heterologous gene expression. We further demonstrate the applicability of these insights using an accessible experimentation system using glass-bead transformation and reestablishment of photosynthesis using psbH mutants, focusing on the mitigation of transcriptional readthrough effects. By characterizing heterologous expression using regulatory elements such as PrrnS, 5'atpA, and 3' rbcL in a sense-transcriptional context, we further documented up to twofold improvement in fluorescence levels. Our findings contribute new tools for molecular biology research in the chloroplast and evidence fundamental gene regulation processes that could enable the development of more effective chloroplast engineering strategies. This work not only paves the way for more efficient genetic engineering of chloroplasts but also deepens our understanding of the regulatory mechanisms at play.

Direct observation correlates NFκB cRel in B cells with activating and terminating their proliferative program.

Antibody responses require the proliferative expansion of B cells controlled by affinity-dependent signals. Yet, proliferative bursts are heterogeneous, varying between 0 and 8 divisions in response to the same stimulus. NFκB cRel is activated in response to immune stimulation in B cells and is genetically required for proliferation. Here, we asked whether proliferative heterogeneity is controlled by natural variations in cRel abundance. We developed a fluorescent reporter mTFP1-cRel for the direct observation of cRel in live proliferating B cells. We found that cRel is heterogeneously distributed among naïve B cells, which are enriched for high expressors in a heavy-tailed distribution. We found that high cRel expressors show faster activation of the proliferative program, but do not sustain it well, with population expansion decaying earlier. With a mathematical model of the molecular network, we showed that cRel heterogeneity arises from balancing positive feedback by autoregulation and negative feedback by its inhibitor IκBε, confirmed by mouse knockouts. Using live-cell fluorescence microscopy, we showed that increased cRel primes B cells for early proliferation via higher basal expression of the cell cycle driver cMyc. However, peak cMyc induction amplitude is constrained by incoherent feedforward regulation, decoding the fold change of cRel activity to terminate the proliferative burst. This results in a complex nonlinear, nonmonotonic relationship between cRel expression and the extent of proliferation. These findings emphasize the importance of direct observational studies to complement gene knockout results and to learn about quantitative relationships between biological processes and their key regulators in the context of natural variations.

Exploiting non-permissive CHO cells as a rapid and efficient method for recombinant HSV-1 isolation.

Using herpes simplex virus type 1 (HSV-1) as a therapeutic tool has recently emerged as a promising strategy for enhancing the treatment of various cancers, particularly those associated with the nervous system, which is the virus's natural site of infection. These viruses are specifically engineered to infect and eradicate tumor cells while leaving healthy cells unharmed. To introduce targeted mutations in specific viral genes, gene-modification techniques such as shuttle vector homologous recombination are commonly employed. Plaque purification is then utilized to select and purify the recombinant virus from the parental viruses. However, plaque purification becomes problematic when the insertion of the desired gene at the target site hampers progeny virus replication, resulting in a lower titer of cell-released virus than the parental virus. This necessitates a laborious initial screening process using approximately 10-15 tissue culture dishes (10 cm), making plaque purification time-consuming and demanding. Although the recently developed CRISPR-Cas9 system significantly enhances the efficiency of homologous integration and editing precision in viral genes, the purification of recombinant variants remains a tedious task. In this study, we propose a rapid and innovative method that employs non-permissive Chinese hamster ovary (CHO) cells, representing a remarkable improvement over the aforementioned arduous process. With this approach, only 1-2 rounds of plaque purification are required. Our proposed protocol demonstrates great potential as a viable alternative to current methods for isolating and purifying recombinant HSV-1 variants expressing fluorescent reporter genes using CHO cells and plaque assays.

Improved protein splicing through viral passaging.

Intervening proteins (inteins) are translated as subdomains within host proteins and removed through an intein-driven splicing reaction where the flanking sequences (exteins) are joined with a peptide bond. Previously, we developed a self-removing translation reporter for labeling Ebola virus (EBOV). In this reporter, an intein (RadA) containing the fluorescent protein ZsGreen (ZsG) is inserted within the EBOV protein VP30. Upon VP30-RadA-ZsG expression from the viral genome, RadA-ZsG is removed from VP30 through the protein splicing activity of RadA, generating functional, non-tagged VP30 and functional ZsGreen. While incorporation of our VP30-RadA-ZsG fusion reporter into recombinant EBOV (rEBOV-RadA-ZsG) resulted in an infectious virus that expresses ZsG upon infection of cells, this virus displayed a replication defect compared to wild-type EBOV, which might be the result of insufficient RadA splicing. Here, we demonstrate that the serial passaging of rEBOV-RadA-ZsG in human cells led to an increase in replication efficiency compared to unpassaged rEBOV-RadA-ZsG. Sequencing of passaged viruses revealed intein-specific mutations. These mutations improve intein activity in both prokaryotic and eukaryotic systems, as well as in multiple extein contexts. Taken together, our findings offer a novel means to select for inteins with enhanced catalytic properties that appear independent of extein context and expression system.IMPORTANCEIntervening proteins (inteins) are self-removing protein elements that have been utilized to develop a variety of innovative protein engineering technologies. Here, we report the isolation of inteins with improved catalytic activity through viral passaging. Specifically, we inserted a highly active intein within an essential protein of Ebola virus and serially passaged this recombinant virus, which led to intein-specific hyper-activity mutations. The identified mutations showed improved intein activity within both bacterial and eukaryotic expression systems and in multiple extein contexts. These results present a new strategy for developing inteins with improved splicing activity.

Characterization of activating cis-regulatory elements from the histone genes of Chlamydomonas reinhardtii.

Regulation of gene expression in eukaryotes is controlled by cis-regulatory modules (CRMs). A major class of CRMs are enhancers which are composed of activating cis-regulatory elements (CREs) responsible for upregulating transcription. To date, most enhancers and activating CREs have been studied in angiosperms; in contrast, our knowledge about these key regulators of gene expression in green algae is limited. In this study, we aimed at characterizing putative activating CREs/CRMs from the histone genes of the unicellular model alga Chlamydomonas reinhardtii. To test the activity of four candidates, reporter constructs consisting of a tetramerized CRE, an established promoter, and a gene for the mCerulean3 fluorescent protein were incorporated into the nuclear genome of C. reinhardtii, and their activity was quantified by flow cytometry. Two tested candidates, Eupstr and Ehist cons, significantly upregulated gene expression and were characterized in detail. Eupstr, which originates from highly expressed genes of C. reinhardtii, is an orientation-independent CRE capable of activating both the RBCS2 and ß2-tubulin promoters. Ehist cons, which is a CRM from histone genes of angiosperms, upregulates the ß2-tubulin promoter in C. reinhardtii over a distance of at least 1.5 kb. The octamer motif present in Ehist cons was identified in C. reinhardtii and the related green algae Chlamydomonas incerta, Chlamydomonas schloesseri, and Edaphochlamys debaryana, demonstrating its high evolutionary conservation. The results of this investigation expand our knowledge about the regulation of gene expression in green algae. Furthermore, the characterized activating CREs/CRMs can be applied as valuable genetic tools.

Mapping the Fate of Hypoxic Cells Using an Irreversible Fluorescent Switch.

Hypoxia has been reported to promote tumor progression and metastasis in murine models, and patients with hypoxic tumors have a worse prognosis. Besides its effect on cancer, normal processes like embryogenesis, or other pathologies such as ischemia, depend on hypoxia-regulated mechanisms. Given the degradable nature of HIF-1/2α in the presence of oxygen, defining the role of hypoxia in modeling biological processes becomes challenging when a cell enters oxygen-rich regions within a tissue. Here, we describe a unique approach to permanently mark cells that experience hypoxia with a fluorescent protein switch that is maintained even after a cell is reoxygenated. This method consists of a dual-viral delivery system that can be transduced into any mammalian cell line.

Turn-on RNA Mango Beacons for trans-acting fluorogenic nucleic acid detection.

The Mango I and II RNA aptamers have been widely used in vivo and in vitro as genetically encodable fluorogenic markers that undergo large increases in fluorescence upon binding to their ligand, TO1-Biotin. However, while studying nucleic acid sequences, it is often desirable to have trans-acting probes that induce fluorescence upon binding to a target sequence. Here, we rationally design three types of light-up RNA Mango Beacons based on a minimized Mango core that induces fluorescence upon binding to a target RNA strand. Our first design is bimolecular in nature and uses a DNA inhibition strand to prevent folding of the Mango aptamer core until binding to a target RNA. Our second design is unimolecular in nature, and features hybridization arms flanking the core that inhibit G-quadruplex folding until refolding is triggered by binding to a target RNA strand. Our third design builds upon this structure, and incorporates a self-inhibiting domain into one of the flanking arms that deliberately binds to, and precludes folding of, the aptamer core until a target is bound. This design separates G-quadruplex folding inhibition and RNA target hybridization into separate modules, enabling a more universal unimolecular beacon design. All three Mango Beacons feature high contrasts and low costs when compared to conventional molecular beacons, with excellent potential for in vitro and in vivo applications.

Dual-Fluorescence Chromosome-Located Labeling System for Accurate In Vivo Single-Cell Gene Expression Analysis in Pseudomonas syringae.

Epigenetic regulation as a means for bacterial adaptation is receiving increasing interest in the last decade. Significant efforts have been directed towards understanding the mechanisms giving raise to phenotypic heterogeneity within bacterial populations and its adaptive relevance. Phenotypic heterogeneity mostly refers to phenotypic variation not linked to genetic differences nor to environmental stimuli. Recent findings on the relevance of phenotypic heterogeneity on some bacterial complex traits are causing a shift from traditional assays where bacterial phenotypes are defined by averaging population-level data, to single-cell analysis that focus on bacterial individual behavior within the population. Fluorescent labeling is a key asset for single-cell gene expression analysis using flow cytometry, fluorescence microscopy, and/or microfluidics.We previously described the generation of chromosome-located transcriptional gene fusions to fluorescent reporter genes using the model bacterial plant pathogen Pseudomonas syringae. These fusions allow researchers to follow variation in expression of the gene(s) of interest, without affecting gene function. In this report, we improve the analytic power of the method by combining such transcriptional fusions with constitutively expressed compatible fluorescent reporter genes integrated in a second, neutral locus of the bacterial chromosome. Constitutively expressed fluorescent reporters allow for the detection of all bacteria comprising a heterogeneous population, regardless of the level of expression of the concurrently monitored gene of interest, thus avoiding the traditional use of stains often incompatible with samples from complex contexts such as the leaf.

Development of a Cre-recombination-based color-switching reporter system for cell fusion detection.

Cell fusion plays a key role in the development and formation of tissues and organs in several organisms. Skeletal myogenesis is assessed in vitro by cell shape and gene and protein expression using immunofluorescence and immunoblotting assays. However, these conventional methods are complex and do not allow for easy time-course observation in living cells. Therefore, this study aimed to develop a Cre recombination-based fluorescent reporter system to monitor cell-cell fusion. We combined green and red fluorescent proteins with a Cre-loxP system to detect syncytium formation using a fluorescent binary switch. This allowed us to visualize mononucleated cells with green fluorescence before fusion and multinucleated syncytia with red fluorescence by conditional expression after cell fusion. The formation of multinuclear myotubes during myogenic differentiation was detected by the change in fluorescence from green to red after Cre-mediated recombination. The distribution of the fluorescence signal correlated with the expression of myogenic differentiation markers. Moreover, red reporter fluorescence intensity was correlated with the number of nuclei contained in the red fluorescent-positive myotubes. We also successfully demonstrated that our fusion monitoring system is applicable to the formation of skeletal muscle myotube and placental syncytiotrophoblast. These results suggest that the color-switching fluorescent reporter system, using Cre-mediated recombination, could be a robust tool used to facilitate the study of cell-to-cell fusion.

Generation and Characterization of an Influenza D Reporter Virus.

Influenza D virus (IDV) can infect various livestock animals, such as cattle, swine, and small ruminants, and was shown to have zoonotic potential. Therefore, it is important to identify viral factors involved in the broad host tropism and identify potential antiviral compounds that can inhibit IDV infection. Recombinant reporter viruses provide powerful tools for studying viral infections and antiviral drug discovery. Here we present the generation of a fluorescent reporter IDV using our previously established reverse genetic system for IDV. The mNeonGreen (mNG) fluorescent reporter gene was incorporated into the IDV non-structural gene segment as a fusion protein with the viral NS1 or NS2 proteins, or as a separate protein flanked by two autoproteolytic cleavage sites. We demonstrate that only recombinant reporter viruses expressing mNG as an additional separate protein or as an N-terminal fusion protein with NS1 could be rescued, albeit attenuated, compared to the parental reverse genetic clone. Serial passaging experiments demonstrated that the mNG gene is stably integrated for up to three passages, after which internal deletions accumulate. We conducted a proof-of-principle antiviral screening with the established fluorescent reporter viruses and identified two compounds influencing IDV infection. These results demonstrate that the newly established recombinant IDV reporter virus can be applied for antiviral drug discovery and monitoring viral replication, adding a new molecular tool for investigating IDV.

Red Fluorescent Protein Variant with a Dual-Peak Emission of Fluorescence.

The marine environment is a rich reservoir of diverse biological entities, many of which possess unique properties that are of immense value to biotechnological applications. One such example is the red fluorescent protein derived from the coral Discosoma sp. This protein, encoded by the DsRed gene, has been the subject of extensive research due to its potential applications in various fields. In the study, a variant of the red fluorescent protein was generated through random mutagenesis using the DsRed2 gene as a template. The process employed error-prone PCR (epPCR) to introduce random mutations, leading to the isolation of twelve gene variants. Among these, one variant stood out due to its unique spectral properties, exhibiting dual fluorescence emission at both 480 nm (green) and 550 nm (red). This novel variant was expressed in both Escherichia coli and zebrafish (Danio rerio) muscle, confirming the dual fluorescence emission in both model systems. One of the immediate applications of this novel protein variant is in ornamental aquaculture. The dual fluorescence can serve as a unique marker or trait, enhancing the aesthetic appeal of aquatic species in ornamental settings.

Episodic live imaging of cone photoreceptor maturation in GNAT2-EGFP retinal organoids.

Fluorescent reporter pluripotent stem cell-derived retinal organoids are powerful tools to investigate cell type-specific development and disease phenotypes. When combined with live imaging, they enable direct and repeated observation of cell behaviors within a developing retinal tissue. Here, we generated a human cone photoreceptor reporter line by CRISPR/Cas9 genome editing of WTC11-mTagRFPT-LMNB1 human induced pluripotent stem cells (iPSCs) by inserting enhanced green fluorescent protein (EGFP) coding sequences and a 2A self-cleaving peptide at the N-terminus of guanine nucleotide-binding protein subunit alpha transducin 2 (GNAT2). In retinal organoids generated from these iPSCs, the GNAT2-EGFP alleles robustly and exclusively labeled immature and mature cones. Episodic confocal live imaging of hydrogel immobilized retinal organoids allowed tracking of the morphological maturation of individual cones for >18 weeks and revealed inner segment accumulation of mitochondria and growth at 12.2 µm3 per day from day 126 to day 153. Immobilized GNAT2-EGFP cone reporter organoids provide a valuable tool for investigating human cone development and disease.

A fluorescent reporter model for the visualization and characterization of TDC.

TDC are hematopoietic cells that combine dendritic cell (DC) and conventional T-cell markers and functional properties. They were identified in secondary lymphoid organs (SLOs) of naïve mice as cells expressing CD11c, major histocompatibility molecules (MHC)-II, and the T-cell receptor (TCR). Despite thorough characterization, a physiological role for TDC remains to be determined. Unfortunately, using CD11c as a marker for TDC has the caveat of its upregulation on different cells, including T cells, upon activation. Here, we took advantage of Zbtb46-GFP reporter mice to explore the frequency and localization of TDC in different tissues at steady state and upon viral infection. RNA sequencing analysis confirmed that TDC sorted from Zbtb46-GFP mice have a gene signature that is distinct from conventional T cells and DC. In addition, this reporter model allowed for identification of TDC in situ not only in SLOs but also in the liver and lung of naïve mice. Interestingly, we found that TDC numbers in the SLOs increased upon viral infection, suggesting that TDC might play a role during viral infections. In conclusion, we propose a visualization strategy that might shed light on the physiological role of TDC in several pathological contexts, including infection and cancer.

A simple, rapid fluorescent reporter-based method for detection of ectopic cre recombinase expression in presumed retinal cell type-targeted mouse lines.

Although cell type-specific Cre recombinase-expressing mouse lines are commonly used to generate conditional knockout of genes of interest, germline recombination and ectopic "leakiness" in Cre recombinase expression in non-specific cell types has been observed in several neuronal and glial-specific Cre lines. This often leads to inadvertent loss of conditional mouse lines, requiring rederivation. It is therefore imperative to be able to monitor and validate cell type-specific Cre recombinase-mediated gene editing. Herein, we describe a simple, inexpensive, rapid ZsGreen fluor-reporter-based strategy for genotype-free identification of ectopic leakiness using a custom-designed, 3-D blue LED light box. We assessed cell type-specific expression in several allegedly specific Cre recombinase mouse lines commonly used in vision research: retinal pigment epithelium (RPE)-specific (VMD2 (Best1) Cre, RPE65 Cre); astrocyte-specific (GFAP Cre); as well as photoreceptor-bipolar progenitor cell-specific (CRX Cre). Our standardized workflow allows facile, rapid identification of ectopic and non-specific Cre recombinase expression in any presume specific Cre mouse line, without the need for genotyping and without causing animal distress.

HCS-Splice: A High-Content Screening Method to Advance the Discovery of RNA Splicing-Modulating Therapeutics.

Nucleic acid therapeutics have demonstrated an impressive acceleration in recent years. They work through multiple mechanisms of action, including the downregulation of gene expression and the modulation of RNA splicing. While several drugs based on the former mechanism have been approved, few target the latter, despite the promise of RNA splicing modulation. To improve our ability to discover novel RNA splicing-modulating therapies, we developed HCS-Splice, a robust cell-based High-Content Screening (HCS) assay. By implementing the use of a two-colour (GFP/RFP) fluorescent splicing reporter plasmid, we developed a versatile, effective, rapid, and robust high-throughput strategy for the identification of potent splicing-modulating molecules. The HCS-Splice strategy can also be used to functionally confirm splicing mutations in human genetic disorders or to screen drug candidates. As a proof-of-concept, we introduced a dementia-related splice-switching mutation in the Microtubule-Associated Protein Tau (MAPT) exon 10 splicing reporter. We applied HCS-Splice to the wild-type and mutant reporters and measured the functional change in exon 10 inclusion. To demonstrate the applicability of the method in cell-based drug discovery, HCS-Splice was used to evaluate the efficacy of an exon 10-targeting siRNA, which was able to restore the correct alternative splicing balance.