Reporter Gene Expression Research Articles

Dexmedetomidine Suppresses Glutamate-Stimulated Neuronal Injury Via MicroRNA-433/Janus Kinase 2/Signal Transducer and Activator of Transcription 3 Pathway.

Cerebral ischemia has been a hotpot in the prevention and treatment of cerebral ischemia. Dexmedetomidine (Dex) is a new type of highly selective α2 adrenergic receptor agonist with pharmacological properties. Quantitative studies have shown that Dex has a protective effect on glutamate (Glu)-induced neuronal damage. however, its mechanism has not been fully elucidated. The purpose of this study was to explore the underlying molecular mechanism by which Dex ameliorates Glu-induced neuronal injury by regulating miR-433/JAK2/STAT3 axis. A model of neuronal injury was constructed by Glu treatment and intervened with Dex. miRNA expression profiling assay was conducted to screen potential miRNAs affected by Dex. Cell viability, lactate dehydrogenase (LDH) release and apoptosis were detected by MTT assay, LDH kit, and TUNEL staining, respectively. Oxidative stress indicators were assessed by ELISA whereas mitochondrial membrane potential (MMP) was assessed by C11-BODIPY581/591 staining. The targeting relationship between the miR-433 and JAK2 was verified by dual-luciferase reporter assay and gene expression was analyzed by quantitative PCR and Western blot. Glu treatment decreased cell viability and MMP and promoted LDH release, apoptosis and oxidative damage. Glu-induced changes in neurons were reversed after Dex treatment through upregulating the miR-433 expression to block the activation of JAK2/STAT3 pathway. Dex protects against Glu-induced neuronal injury by regulating miR-433/JAK2/STAT3 pathway, which provides new insights into the treatment of neuronal injury.

Plant lysin motif extracellular proteins are required for arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizal fungi (AMF) can form a mutually beneficial symbiotic relationship with most land plants. They are known to secrete lysin motif (LysM) effectors into host root cells for successful colonization. Intriguingly, plants secrete similar types of LysM proteins; however, their role in plant-microbe interactions is unknown. Here, we show that Medicago truncatula deploys LysM extracellular (LysMe) proteins to facilitate symbiosis with AMF. Promoter analyses demonstrated that three M. truncatula LysMe genes MtLysMe1/2/3, are expressed in arbuscule-containing cells and those adjacent to intercellular hyphae. Localization studies showed that these proteins are targeted to the periarbuscular space between the periarbuscular membrane and the fungal cell wall of the branched arbuscule. M. truncatula mutants in which MtLysMe2 was knocked out via CRISPR/Cas9-targeted mutagenesis exhibited a significant reduction in AMF colonization and arbuscule formation, whereas genetically complemented transgenic plants restored wild-type level AMF colonization. In addition, knocking out the ortholog of MtLysMe2 in tomato resulted in a similar defect in AMF colonization. In vitro binding affinity precipitation assays suggested binding of MtLysMe1/2/3 with chitin and chitosan, while microscale thermophoresis (MST) assays revealed weak binding of these proteins with chitooligosaccharides. Moreover, application of purified MtLysMe proteins to root segments could suppress chitooctaose (CO8)-induced reactive oxygen species production and expression of reporter genes of the immune response without impairing chitotetraose (CO4)-triggered symbiotic responses. Taken together, our results reveal that plants, like their fungal partners, also secrete LysM proteins to facilitate symbiosis establishment.

Evaluation of the effectiveness of RNA silencing in the filamentous fungus Aspergillus niger using a binary vector integrated with Gateway recombination cloning technology

The filamentous fungus Aspergillus niger is widely employed in the production of citric acid and some industrial enzymes such as phytase, glucoamylase, and glucose oxidase. Currently, several genetic modification tools have been developed and applied to this fungal species. However, the RNA silencing technique is less used for A. niger. In the present study, an RNA silencing binary vector integrated with the Gateway recombination cloning technology was evaluated in A. niger. The integration of Gateway recombination cloning technology in binary vectors for rapid generation of hairpin RNA structure allowed the assessment of inhibition of target gene expression through mRNA degradation. The RNA silencing constructs were successfully transferred into A. nigervia Agrobacterium tumefaciens. Results showed that the RNA silencing vectors were effective in downregulating the expression of the DsRed fluorescent reporter gene and the stuA regulatory gene. Interestingly, the silenced mutants of stuA exhibited a significant reduction in fungal sporulation. This finding revealed that the RNA silencing technique with the examined Gateway binary vector represents a potential genetic tool for functional studies of target genes in A. niger.

Structure elucidation and biological activities of perylenequinones from an Alternaria species

The KEAP1-Nrf2/ARE pathway is a pivotal cytoprotective regulator against oxidative stress which plays an important role in the development of many inflammatory diseases and cancer. Activation of the Nrf2 transcription factor by oxidative stress or electrophiles regulates antioxidant response element (ARE)-dependent transcription of antioxidative, detoxifying, and anti-inflammatory proteins. Therefore, modulators of the KEAP1-Nrf2/ARE pathway have received considerable interest as therapeutics to protect against diseases where oxidative stress constitutes the underlying pathophysiology. In a search for fungal secondary metabolites affecting the Nrf2/ARE-dependent expression of a luciferase reporter gene in BEAS-2B cells, three new perylenequinones, compounds 1, 2, and 3, together with altertoxin-I (ATX-I), were isolated from fermentations of an Alternaria species. The structures of the compounds were elucidated by a combination of one- and two-dimensional NMR spectroscopy and mass spectrometry. Compound 1 and ATX-I exhibited strong cytotoxic effects with LC50-values of 3.8 µM and 6.43 µM, respectively, whereas compound 3 showed no cytotoxic effects up to 100 µM on BEAS-2B cells. ATX-I induced ARE-dependent luciferase expression approximately fivefold and compound 1 approximately 2.6-fold at a concentration of 3 µM in transiently transfected BEAS-2B cells. In addition, compound 1 and ATX-I exhibited strong oxidative effects, whereas compound 3 did not show significant oxidative properties. For compound 1 and ATX-I, a strong upregulation of heme oxygenase-1 could be observed on mRNA and protein level in treated BEAS-2B cells. Moreover, compound 3 significantly decreased sod3 mRNA levels after induction of oxidative stress with benzoquinone.

Nanoparticle‐Decorated Biomimetic Extracellular Matrix for Cell Nanoencapsulation and Regulation

AbstractCell encapsulation has been studied for various applications ranging from cell transplantation to biological production. However, current encapsulation technologies focus on cell protection rather than cell regulation that is essential to most if not all cell‐based applications. Here we report a method for cell nanoencapsulation and regulation using an ultrathin biomimetic extracellular matrix as a cell nanocapsule to carry nanoparticles (CN2). This method allows high‐capacity nanoparticle retention at the vicinity of cell surfaces. The encapsulated cells maintain high viability and normal metabolism. When gold nanoparticles (AuNPs) are used as a model to decorate the nanocapsule, light irradiation transiently increases the temperature, leading to the activation of the heat shock protein 70 (HSP70) promoter and the regulation of reporter gene expression. As the biomimetic nanocapsule can be decorated with any or multiple NPs, CN2 is a promising platform for advancing cell‐based applications.

Nanoparticle-Decorated Biomimetic Extracellular Matrix for Cell Nanoencapsulation and Regulation.

Cell encapsulation has been studied for various applications ranging from cell transplantation to biological production. However, current encapsulation technologies focus on cell protection rather than cell regulation that is essential to most if not all cell-based applications. Here we report a method for cell nanoencapsulation and regulation using an ultrathin biomimetic extracellular matrix as a cell nanocapsule to carry nanoparticles (CN2 ). This method allows high-capacity nanoparticle retention at the vicinity of cell surfaces. The encapsulated cells maintain high viability and normal metabolism. When gold nanoparticles (AuNPs) are used as a model to decorate the nanocapsule, light irradiation transiently increases the temperature, leading to the activation of the heat shock protein 70 (HSP70) promoter and the regulation of reporter gene expression. As the biomimetic nanocapsule can be decorated with any or multiple NPs, CN2 is a promising platform for advancing cell-based applications.

Expression of GFP and DsRed fluorescent proteins after gene electrotransfer of tumour cells in vitro

Fluorescent reporter genes are widely used to study the transfection of various types of primary cells and cell lines. The aim of our research was to investigate the expression dynamics of GFP and DsRed reporter genes individually and combined after gene electrotransfer of plasmids with two different electroporation protocols in B16F10 and CT26 cells in vitro. The cytotoxicity after gene electrotransfer of both plasmids was first determined. Second, the intensity of fluorescence and the percentage of cells transfected with both plasmids individually and in combination were monitored in real time. The results show that the percentage of viability after gene electrotransfer of plasmids using the EP2 pulses was significantly higher compared to the EP1 pulses. In contrast, the percentage of transfected cells and fluorescence intensity were higher after gene electrotransfer with the EP1 pulse protocol. Moreover, the percentage of transfected cells was higher and started earlier in the B16F10 cell line than in the CT26 cell line. However, fluorescence intensity was higher in CT26 cells. Co-expression of fluorescent proteins was achieved only in a small number of cells. In conclusion, this study elucidated some of the dynamics of reporter gene expression in cancer cell lines after gene electrotransfer.

Chromosomal engineering of inducible isopropanol- butanol-ethanol production in Clostridium acetobutylicum.

The use of environmentally damaging petrochemical feedstocks can be displaced by fermentation processes based on engineered microbial chassis that recycle biomass-derived carbon into chemicals and fuels. The stable retention of introduced genes, designed to extend product range and/or increase productivity, is essential. Accordingly, we have created multiply marked auxotrophic strains of Clostridium acetobutylicum that provide distinct loci (pyrE, argH, purD, pheA) at which heterologous genes can be rapidly integrated using allele-coupled exchange (ACE). For each locus, ACE-mediated insertion is conveniently selected on the basis of the restoration of prototrophy on minimal media. The Clostridioides difficile gene (tcdR) encoding an orthogonal sigma factor (TcdR) was integrated at the pyrE locus under the control of the lactose-inducible, bgaR::PbgaL promoter to allow the simultaneous control of genes/operons inserted at other disparate loci (purD and pheA) that had been placed under the control of the PtcdB promoter. In control experiments, dose-dependent expression of a catP reporter gene was observed with increasing lactose concentration. At the highest doses tested (10mM) the level of expression was over 10-fold higher than if catP was placed directly under the control of bgaR::PbgaL and over 2-fold greater than achieved using the strong Pfdx promoter of the Clostridium sporogenes ferredoxin gene. The utility of the system was demonstrated in the production of isopropanol by the C. acetobutylicum strain carrying an integrated copy of tcdR following the insertion of a synthetic acetone operon (ctfA/B, adc) at the purD locus and a gene (sadh) encoding a secondary dehydrogenase at pheA. Lactose induction (10mM) resulted in the production of 4.4g/L isopropanol and 19.8g/L Isopropanol-Butanol-Ethanol mixture.

DIPG-21. LINKING DETERMINATION OF CELL FATE IN THE DEVELOPING CENTRAL NERVOUS SYSTEM TO DIFFUSE MIDLINE GLIOMA

Abstract Diffuse midline glioma, K27-altered (DMG) is a treatment resistant, uniformly fatal cancer that arises predominantly in children. Next-generation sequencing has revealed that the majority of patients with DMG harbor K27M mutations in canonical histone variants H3.1 and H3.3, leading to global epigenetic dysregulation and an oligodendrocyte precursor cell (OPC) transcriptional profile. DLX2 is a homeobox transcription factor that directly represses OPC cell fate and promotes a GABAergic interneuron cell fate during neurogenesis. We identified DLX2 occupancy of early (Olig2, Nkx2.2) and late (Myt1, Plp1) genes required in OPC differentiation in vivo. Co-expression of DLX2 to target sequences reduced reporter gene expression in vitro. The Dlx1/Dlx2 double knockout (DKO) mouse showed increases in Olig2, Nkx2.2, and Plp-1 expression in vivo. Transient over-expression of a Dlx2-GFP construct into murine DMG cells resulted in significant increased expression of Gad1/2 isoforms and decreased Olig2 and Nkx2.2, global restoration of H3K27me3, as well as a reduction in migration, invasion, and colony formation in vitro. An RNAseq database containing 45 human DMG cell lines showed an inverse correlation between DLX2 expression and OLIG1/2. Cell fate changes in DLX2high/OLIG1/2low cell lines were evaluated by knockout of DLX2 using CRISPR/Cas9. Concurrently, OLIG1/2high/DLX2low expressing cell lines underwent stable transfection of an DLX2-mCherry overexpression construct. Successful alteration of DLX2 expression was confirmed by qPCR and immunoblot in knockout and overexpressing human DMG cell lines. Analysis of histone H3 marks (K27me3, K27ac), K27M, and OPC signature genes will be presented along with comparison of altered OPC genes from the Dlx1/Dlx2 DKO mouse as determined by RNAseq. The manipulation of DLX2 expression in human DMG cells both in vitro and in vivo provides a model system for assessing the contribution of a homeobox transcription factor required for neurogenesis to cell fate regulation and effects on tumorigenicity in DMG.

BIC2, a Cryptochrome Function Inhibitor, Is Involved in the Regulation of ABA Responses in Arabidopsis.

The plant hormone ABA (abscisic acid) is able to regulate plant responses to abiotic stresses via regulating the expression of ABA response genes. BIC1 (Blue-light Inhibitor of Cryptochromes 1) and BIC2 have been identified as the inhibitors of plant cryptochrome functions, and are involved in the regulation of plant development and metabolism in Arabidopsis . In this study, we report the identification of BIC2 as a regulator of ABA responses in Arabidopsis . RT-PCR (Reverse Transcription-Polymerase Chain Reaction) results show that the expression level of BIC1 remained largely unchanged, but that of BIC2 increased significantly in response to ABA treatment. Transfection assays in Arabidopsis protoplasts show that both BIC1 and BIC2 were mainly localized in the nucleus, and were able to activate the expression of the co-transfected reporter gene. Results in seed germination and seedling greening assays show that ABA sensitivity was increased in the transgenic plants overexpressing BIC2, but increased slightly, if any, in the transgenic plants overexpressing BIC1. ABA sensitivity was also increased in the bic2 single mutants in seedling greening assays, but no further increase was observed in the bic1 bic2 double mutants. On the other hand, in root elongation assays, ABA sensitivity was decreased in the transgenic plants overexpressing BIC2, as well as the bic2 single mutants, but no further decrease was observed in the bic1 bic2 double mutants. By using qRT-PCR (quantitative RT-PCR), we further examined how BIC2 may regulate ABA responses in Arabidopsis , and found that inhibition of ABA on the expression of the ABA receptor genes PYL4 (PYR1-Like 4) and PYL5 were decreased, but promotion of ABA on the expression of the protein kinase gene SnRK2.6 (SNF1-Related Protein Kinases 2.6) was enhanced in both the bic1 bic2 double mutants and 35S:BIC2 overexpression transgenic plants. Taken together, our results suggest that BIC2 regulates ABA responses in Arabidopsis possibly by affecting the expression of ABA signaling key regulator genes.

Deep learning model with L1 penalty for predicting breast cancer metastasis using gene expression data

Breast cancer has the highest incidence and death rate among women; moreover, its metastasis to other organs increases the mortality rate. Since several studies have reported gene expression and cancer prognosis to be related, the study of breast cancer metastasis using gene expression is crucial. To this end, a novel deep neural network architecture, deep learning-based cancer metastasis estimator (DeepCME), is proposed in this paper for predicting breast cancer metastasis. However, the problem of overfitting occurs frequently while training deep learning models using gene expression data because they contain a large number of genes and the sample size is rather small. To address overfitting, several regularization methods are implemented, such as L1 penalty, batch normalization, and dropout. To demonstrate the superior performance of our model, area under curve (AUC) scores are evaluated and then compared with five baseline models: logistic regression, support vector classifier (SVC), random forest, decision tree, and k-nearest neighbor. Considering results, DeepCME demonstrates the highest average AUC scores in most cross-validation cases, and the average AUC score of DeepCME is 0.754, which is approximately 12.9% higher than SVC, the second-best model. In addition, the 30 most significant genes related to breast cancer metastasis are identified based on DeepCME results and some are discussed in further detail considering the reports from some previous medical studies. Considering the high expense involved in measuring the expression of a single gene, the ability to develop the cost-effective and time-efficient tests using only a few key genes is valuable. Based on this study, we expect DeepCME to be utilized clinically for predicting breast cancer metastasis and be applied to other types of cancer as well after further research.

Seeing red: a modified RUBY reporter assay for visualizing inplanta protein-protein interactions.

Seeing red: a modified RUBY reporter assay for visualizing inplanta protein-protein interactions.

Characterizing the tumor selectivity of oncolytic adenovirus ICVB-1042: Analysis of S-phase induction, viral replication, and cytolytic activity in human primary normal cell types.

e14503 Background: Adenoviral-based oncolytic virus (OV) therapeutics include mutations that enable activity in tumor cells and limit toxicity in normal cells. These tumor selectivity approaches often involve mutating viral E1A function or expression; however, such modifications allow viral activity in normal cells or limit anti-tumor potency. ICVB-1042 was engineered with dual modifications in E1A and E4orf6/7, resulting in superior tumor selectivity that does not compromise lytic potency in tumor cells. This improved tumor selective virus minimizes off-tumor activity and promotes robust tumor lysis. Methods: ICVB-1042 was compared to viruses with wildtype E1A and E4orf6/7 and a clinically tested virus with an E1A mutation (ICVB-2006). Viral function and killing were assessed in primary normal human renal (HRE) and bronchial (BrE) epithelial cells in the presence of the inhibitor palbociclib to pharmacologically block S-phase entry via host cell signaling. HRE and BrE were infected with virus and evaluated for indicators of viral toxicity in normal cells. Viral replication was quantified by droplet digital polymerase chain reaction (ddPCR); cell lysis was quantified by WST-1 assay. An S-phase induction assay was developed using an mCherry-Geminin reporter that allows imaging-based quantification of viral-dependent entry to S-phase. Viral activity in cells was compared with and without the CDK4/6 inhibitor using a viral-encoded fluorescent reporter to determine the requirement for host cell proliferation on viral function. Results: ICVB-1042 demonstrated superior protection in normal cells to comparator ICVB-2006 and viruses lacking tumor selectivity modifications. Palbociclib steeply reduced viral reporter gene expression (63-88% reduction), viral replication ( > 10-fold reduction), and cytolytic activity of ICVB-1042, while non-selective viruses were unaffected by this treatment. Non-selective virus infection resulted in 3.3- to 6.1-fold higher levels of S-phase entry compared to ICVB-1042. ICVB-2006 infection resembled non-selective viruses at the level of S-phase induction (3.1- to 3.3-fold above ICVB-1042) and viral reporter gene expression was modestly inhibited by palbociclib (12-31% reduction). Conclusions: The improved tumor selectivity of ICVB-1042 results in protection of normal, non-proliferative cells from viral-mediated toxicity. The conventional approach of ICVB-2006 with a single E1A mutation leaves viral replication and cell death in normal human cells mostly intact. Together, these observations support that ICVB-1042 presents normal cell protection and reduced toxicity risks associated with OV therapy.

Accelerated Evolution Analysis Uncovers PKNOX2 as a Key Transcription Factor in the Mammalian Cochlea.

The genetic bases underlying the evolution of morphological and functional innovations of the mammalian inner ear are poorly understood. Gene regulatory regions are thought to play an important role in the evolution of form and function. To uncover crucial hearing genes whose regulatory machinery evolved specifically in mammalian lineages, we mapped accelerated noncoding elements (ANCEs) in inner ear transcription factor (TF) genes and found that PKNOX2 harbors the largest number of ANCEs within its transcriptional unit. Using reporter gene expression assays in transgenic zebrafish, we determined that four PKNOX2-ANCEs drive differential expression patterns when compared with ortholog sequences from close outgroup species. Because the functional role of PKNOX2 in cochlear hair cells has not been previously investigated, we decided to study Pknox2 null mice generated by CRISPR/Cas9 technology. We found that Pknox2-/- mice exhibit reduced distortion product otoacoustic emissions (DPOAEs) and auditory brainstem response (ABR) thresholds at high frequencies together with an increase in peak 1 amplitude, consistent with a higher number of inner hair cells (IHCs)-auditory nerve synapsis observed at the cochlear basal region. A comparative cochlear transcriptomic analysis of Pknox2-/- and Pknox2+/+ mice revealed that key auditory genes are under Pknox2 control. Hence, we report that PKNOX2 plays a critical role in cochlear sensitivity at higher frequencies and that its transcriptional regulation underwent lineage-specific evolution in mammals. Our results provide novel insights about the contribution of PKNOX2 to normal auditory function and to the evolution of high-frequency hearing in mammals.

Radiovirotherapy at twenty

Radiovirotherapy at twenty

Plp1 in the enteric nervous system is preferentially expressed during early postnatal development in mouse as DM20, whose expression appears reliant on an intronic enhancer.

Recently, the myelin proteolipid protein gene (Plp1) was shown to be expressed in the glia of the enteric nervous system (ENS) in mouse. However, beyond this, not much is known about its expression in the intestine. To address this matter, we investigated Plp1 expression at the mRNA and protein levels in the intestine of mice at different ages (postnatal days 2, 9, 21, and 88). In this study, we show that Plp1 expression preferentially occurs during early postnatal development, primarily as the DM20 isoform. Western blot analysis indicated that DM20 migrated according to its formula weight when isolated from the intestine. However, mobilities of both PLP and DM20 were faster than expected when procured from the brain. The 6.2hPLP(+)Z/FL transgene, which uses the first half of the human PLP1 gene to drive expression of a lacZ reporter gene, recapitulated the developmental pattern observed with the native gene in the intestine, indicating that it can be used as a proxy for Plp1 gene expression. As such, the relative levels of β-galactosidase (β-gal) activity emanating from the 6.2hPLP(+)Z/FL transgene suggest that Plp1 expression is highest in the duodenum, and decreases successively along the segments, toward the colon. Moreover, removal of the wmN1 enhancer region from the transgene (located within Plp1 intron 1) resulted in a dramatic reduction in both transgene mRNA levels and β-gal activity in the intestine, throughout development, suggesting that this region contains a regulatory element crucial for Plp1 expression. This is consistent with earlier studies in both the central and peripheral nervous systems, indicating that it may be a common (if not universal) means by which Plp1 gene expression is governed.

Axonal T3 uptake and transport can trigger thyroid hormone signaling in the brain.

The development of the brain, as well as mood and cognitive functions, are affected by thyroid hormone (TH) signaling. Neurons are the critical cellular target for TH action, with T3 regulating the expression of important neuronal gene sets. However, the steps involved in T3 signaling remain poorly known given that neurons express high levels of type 3 deiodinase (D3), which inactivates both T4 and T3. To investigate this mechanism, we used a compartmentalized microfluid device and identified a novel neuronal pathway of T3 transport and action that involves axonal T3 uptake into clathrin-dependent, endosomal/non-degradative lysosomes (NDLs). NDLs-containing T3 are retrogradely transported via microtubules, delivering T3 to the cell nucleus, and doubling the expression of a T3-responsive reporter gene. The NDLs also contain the monocarboxylate transporter 8 (Mct8) and D3, which transport and inactivate T3, respectively. Notwithstanding, T3 gets away from degradation because D3's active center is in the cytosol. Moreover, we used a unique mouse system to show that T3 implanted in specific brain areas can trigger selective signaling in distant locations, as far as the contralateral hemisphere. These findings provide a pathway for L-T3 to reach neurons and resolve the paradox of T3 signaling in the brain amid high D3 activity.

Pou4f1-Tbr1 transcriptional cascade controls the formation of Jam2-expressing retinal ganglion cells.

More than 40 retinal ganglion cell (RGC) subtypes have been categorized in mouse based on their morphologies, functions, and molecular features. Among these diverse subtypes, orientation-selective Jam2-expressing RGCs (J-RGCs) has two unique morphologic characteristics: the ventral-facing dendritic arbor and the OFF-sublaminae stratified terminal dendrites in the inner plexiform layer. Previously, we have discovered that T-box transcription factor T-brain 1 (Tbr1) is expressed in J-RGCs. We further found that Tbr1 is essential for the expression of Jam2, and Tbr1 regulates the formation and the dendritic morphogenesis of J-RGCs. However, Tbr1 begins to express in terminally differentiated RGCs around perinatal stage, suggesting that it is unlikely involved in the initial fate determination for J-RGC and other upstream transcription factors must control Tbr1 expression and J-RGC formation. Using the Cleavage Under Targets and Tagmentation technique, we discovered that Pou4f1 binds to Tbr1 on the evolutionary conserved exon 6 and an intergenic region downstream of the 3'UTR, and on a region flanking the promoter and the first exon of Jam2. We showed that Pou4f1 is required for the expression of Tbr1 and Jam2, indicating Pou4f1 as a direct upstream regulator of Tbr1 and Jam2. Most interestingly, the Pou4f1-bound element in exon 6 of Tbr1 possesses high-level enhancer activity, capable of directing reporter gene expression in J-RGCs. Together, these data revealed a Pou4f1-Tbr1-Jam2 genetic hierarchy as a critical pathway in the formation of J-RGC subtype.

Strong ubiquitous micro-promoters for recombinant adeno-associated viral vectors

Significant progress has been made in developing recombinant adeno-associated virus (rAAV) for clinical gene therapy. While rAAV is a versatile gene delivery platform, its packaging limit of 4.7 kb limits the diseases it can target. Here, we report two unusually small promoters that enable the expression of larger transgenes than standard promoters. These micro-promoters are only 84 (MP-84) and 135bp (MP-135) in size but have activity in most cells and tissues comparable to the CAG promoter, the strongest ubiquitous promoter to date. MP-84- and MP-135-based rAAV constructs displayed robust activity in cultured cells from the three different germ-layer lineages. In addition, reporter gene expression was documented in human primary hepatocytes and pancreatic islets and in multiple mouse tissues invivo, including brain and skeletal muscle. MP-84 and MP-135 will enable the therapeutic expression of transgenes currently too large for rAAV vectors.

Retro-Tango enables versatile retrograde circuit tracing in Drosophila.

Transsynaptic tracing methods are crucial tools in studying neural circuits. Although a couple of anterograde tracing methods and a targeted retrograde tool have been developed in Drosophila melanogaster, there is still need for an unbiased, user-friendly, and flexible retrograde tracing system. Here, we describe retro-Tango, a method for transsynaptic, retrograde circuit tracing and manipulation in Drosophila. In this genetically encoded system, a ligand-receptor interaction at the synapse triggers an intracellular signaling cascade that results in reporter gene expression in presynaptic neurons. Importantly, panneuronal expression of the elements of the cascade renders this method versatile, enabling its use not only to test hypotheses but also to generate them. We validate retro-Tango in various circuits and benchmark it by comparing our findings with the electron microscopy reconstruction of the Drosophila hemibrain. Our experiments establish retro-Tango as a key method for circuit tracing in neuroscience research.