EGFP Reporter Research Articles

Bone Marrow Sinusoidal Endothelial Cells Are a Site of Fgf23 Upregulation in Iron Deficiency Anemia

Iron deficiency anemia (IDA) has been identified as a potent stimulator of FGF23 (fibroblast growth factor 23), a phosphaturic hormone classically thought to be produced by bone-embedded osteocytes. Recently, both phlebotomy and erythropoietin administration have been shown to upregulate FGF23 production in bone marrow. However, the cell type(s) mediating FGF23 upregulation in states of perturbed erythropoiesis require further clarification.Tmprss6-/- mice exhibit hepcidin elevation leading to systemic iron deficiency and iron-restricted anemia. We previously reported that Tmprss6-/- mice exhibit altered phosphate balance, elevated circulating FGF23, and Fgf23 mRNA upregulation in bone marrow but not cortical bone. Here, we clarify the sites of Fgf23 promoter activity in Tmprss6 -/- bone marrow using a reporter allele in which the enhanced green fluorescent protein (eGFP) coding sequence has been knocked into the endogenous Fgf23 locus. We generated Tmprss6 +/+,Tmprss6 +/-, and Tmprss6 -/- littermates of both sexes that carried either one (Fgf23 +/eGFP) or zero (Fgf23 +/+) copies of the reporter allele. Tmprss6-/- mice showed hyperhepcidinemia, hypoferremia, microcytic anemia, and tissue iron deficiency, which were not altered by heterozygous Fgf23 disruption (Figure 1A-C). By ELISA, Tmprss6-/- Fgf23 +/eGFP mice showed plasma levels of “total” FGF23 (intact, active hormone and C-terminal cleaved fragments) that remained markedly elevated compared to Tmprss6+/+ littermates (Figure 1D). Total FGF23 elevation in Tmprss6-/- Fgf23 +/eGFP mice was slightly less pronounced than Tmprss6-/- Fgf23 +/+ mice, suggesting an effect of Fgf23 gene dosage. In mice with 2 intact Fgf23 alleles, serum erythropoietin showed a strong linear correlation with plasma total FGF23.By confocal imaging, femurs of mice carrying the Fgf23 eGFP allele showed green fluorescence in vascular regions of the bone marrow but not in the bone cortex. Green fluorescence was more intense in Tmprss6-/- Fgf23+/eGFP mice than non-anemic controls. By flow cytometry of enzymatically digested bone marrow, we observed bright green fluorescence in a subset of endothelial cells (CD45 - Ter119 - CD31 +) exclusively in mice carrying the Fgf23 eGFP reporter allele (Figure 1E). The percentage of endothelial cells that were GFP bright was higher in Tmprss6-/- Fgf23 +/eGFP versus non-anemic mice. To clarify the endothelial cell subtype that expresses Fgf23, we mined published transcriptomic datasets from mice of normal iron balance and discovered higher Fgf23 mRNA in bone marrow sinusoidal endothelial cells compared to other bone marrow endothelial cell populations. Accordingly, we used anti-GFP immunohistochemistry in formalin-fixed bone marrow sections to assess Fgf23 eGFP reporter allele expression in the context of tissue architecture. Tmprss6-/- Fgf23 +/eGFP mice showed GFP expression in bone marrow sinusoidal endothelial cells, which was more intense than in non-anemic controls (Figure 1F). GFP reporter expression was also detected in rare cells of the thymus but not in liver, spleen, heart, muscle, or kidney. Collectively, our data reveal that bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in chronic IDA.Because IDA in Tmprss6-/- mice results from pathologic hepcidin elevation, we also sought to determine if bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in anemic mice with intact hepcidin regulation. We therefore subjected Fgf23 +/eGFP mice (with 2 intact Tmprss6 alleles) to a 500µl phlebotomy regimen (with saline volume replacement) known to induce marked anemia and hepcidin suppression. Compared to non-phlebotomized Fgf23 +/eGFP controls, phlebotomized Fgf23 +/eGFP mice showed severe anemia, elevated serum erythropoietin, and elevated plasma FGF23 18 hours after blood loss. Additionally, immunohistochemistry revealed more intense GFP expression in bone marrow sinusoidal endothelial cells of phlebotomized Fgf23 +/eGFP mice than non-phlebotomized controls. Taken together, our results show for the first time that bone marrow sinusoidal endothelial cells are a site of Fgf23 upregulation in both acute and chronic anemia. Given the serum erythropoietin elevation in both models, our findings suggest that erythropoietin may act directly or indirectly on sinusoidal endothelial cells to promote FGF23 production during anemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Unraveling Heterogeneity of Aged Hematopoietic Stem Cells By Single-Cell RNA Sequence Analysis

During aging, hematopoietic stem cells (HSCs) alter quantitatively as well as qualitatively due to accumulating damages induced by intrinsic and extrinsic stresses. Functional decline of HSCs causes deregulated hematopoiesis resulting in anemia, immune dysfunction, and increased risk of hematologic malignancies. The absolute number of HSCs in aged mice evidently increases compared to young mice. In addition, aged HSCs show abnormal hematopoiesis such as myeloid-biased differentiation accompanied by low production of lymphocytes. However, the molecular mechanisms underlying age-associated changes in hematopoiesis remain largely unknown. In this study, we performed single-cell RNA sequence analysis (scRNA-seq) of HSCs from young (10-week-old), middle-aged (12-month-old) and aged (20-month-old) mice to gain insight into the dynamics of HSC aging.scRNA-seq analysis revealed three major clusters (A, B, C) and three minor clusters (D, E, F) in young HSCs. Of interest, aged HSCs also showed similar cluster formation. One of the minor clusters was characterized by gene signature associated with inflammatory response and significantly increased with age, while the remaining two minor clusters, which showed cell cycle gene signature, did not change in proportion. One of the major clusters, Cluster C, which showed the strongest expression of HSC-specific gene sets compared with other clusters, moderately increased with age.Our scRNA-seq analysis confirmed upregulation of age-related genes previously reported, such as Selp, Mt1, and Vwf, in a considerable portion of aged HSCs. von Willebrand factor (Vwf) encodes a blood glycoprotein produced by megakaryocytes and endothelial cells. Several groups have reported that Vwf-expressing HSCs show myeloid/platelet-biased differentiation (Joana C et al., Nature 2013; Sandra P et al., Developmental Cell 2018). Importantly, our scRNA-seq data identified that Clusterin (Clu) is rarely expressed in young HSCs and is dramatically upregulated in aged HSCs. Clu expression was predominantly increased in one of the major clusters, Cluster C. Clusterin encodes a secreted chaperone involved in clearance of cellular debris and regulation of apoptosis. We hypothesized that Clu would be useful as a marker of a unique subpopulation of aged HSCs, and thus conducted further analysis by using Clu reporter mice with Clu BAC clone, in which an EGFP reporter gene was inserted at the initiating ATG codon of the Clu gene so that EGFP expression is driven by the regulatory sequences of the BAC gene.Clu/GFP was preferentially expressed in HSCs and at lower frequencies in MPP1 than HSCs in Lineage -Sca-1 +c-Kit + (LSK) cell fraction. Clu-positive HSCs expressed high levels of CD150 and were detected in 10% and 60% of HSCs in 10-week-old young mice and 8-month-old middle-aged mice, respectively, indicating that Clu-positive HSCs increase with aging. We next assessed the function of Clu-positive HSCs in young mice. Clu-positive young HSCs established significantly lower chimerism than Clu-negative young HSCs and preferentially differentiated into myeloid cells in competitive transplantation assays. RNA-seq analysis of Clu-positive and Clu-negative HSCs from young mice confirmed that Clu-positive HSCs show the gene signature of myeloid-biased HSCs. Characterization of Clu-positive and negative subpopulations in aged HSCs is currently underway. These results suggest that Clu-positive HSCs represent myeloid-biased HSCs which expand with aging, thus Clu expression serves as a novel marker to monitor the alterations in HSC heterogeneity with aging. DisclosuresIwama: Nissan Chemical Corporation: Research Funding.

The concurrent stimulation of Wnt and FGF8 signaling induce differentiation of dental mesenchymal cells into odontoblast-like cells

Fibroblast growth factor 8 (FGF8) is known to be a potent stimulator of canonical Wnt/β-catenin activity, an essential factor for tooth development. In this study, we analyzed the effects of co-administration of FGF8 and a CHIR99021 (GSK3β inhibitor) on differentiation of dental mesenchymal cells into odontoblasts. Utilizing Cre-mediated EGFP reporter mice, dentin matrix protein 1 (Dmp1) expression was examined in mouse neonatal molar tooth germs. At birth, expression of Dmp1-EGFP was not found in mesenchymal cells but rather epithelial cells, after which Dmp1-positive cells gradually emerged in the mesenchymal area along with disappearance in the epithelial area. Primary cultured mesenchymal cells from neonatal tooth germ specimens showed loss of Dmp1-EGFP positive signals, whereas addition of Wnt3a or the CHIR99021 significantly regained Dmp1 positivity within approximately 2 weeks. Other odontoblast markers such as dentin sialophosphoprotein (Dspp) could not be clearly detected. Concurrent stimulation of primary cultured mesenchymal cells with the CHIR99021 and FGF8 resulted in significant upregulation of odonto/osteoblast proteins. Furthermore, increased expression levels of runt-related transcription factor 2 (Runx2), osterix, and osteocalcin were also observed. The present findings indicate that coordinated action of canonical Wnt/β-catenin and FGF8 signals is essential for odontoblast differentiation of tooth germs in mice.

Not All Lectins Are Equally Suitable for Labeling Rodent Vasculature.

The vascular system is vital for all tissues and the interest in its visualization spans many fields. A number of different plant-derived lectins are used for detection of vasculature; however, studies performing direct comparison of the labeling efficacy of different lectins and techniques are lacking. In this study, we compared the labeling efficacy of three lectins: Griffonia simplicifolia isolectin B4 (IB4); wheat germ agglutinin (WGA), and Lycopersicon esculentum agglutinin (LEA). The LEA lectin was identified as being far superior to the IB4 and WGA lectins in histological labeling of blood vessels in brain sections. A similar signal-to-noise ratio was achieved with high concentrations of the WGA lectin injected during intracardial perfusion. Lectins were also suitable for labeling vasculature in other tissues, including spinal cord, dura mater, heart, skeletal muscle, kidney, and liver tissues. In uninjured tissues, the LEA lectin was as accurate as the Tie2–eGFP reporter mice and GLUT-1 immunohistochemistry for labeling the cerebral vasculature, validating its specificity and sensitivity. However, in pathological situations, e.g., in stroke, the sensitivity of the LEA lectin decreases dramatically, limiting its applicability in such studies. This work can be used for selecting the type of lectin and labeling method for various tissues.

Application of Hypoxia-Driven Dual-Reporter Model to Explore the Effect of Hypoxia on Radiosensitivity of Human Nasopharyngeal Carcinoma Cells

<h3>Purpose/Objective(s)</h3> hypoxia has been commonly observed in human nasopharyngeal carcinoma (NPC) which is associated with resistance to radiation and malignant phenotypes. The purpose of the study was to apply our build-in hypoxia-driven dual reporter model to explore the effect of hypoxia on radiosensitivity <b>in vitro</b> and <b>in vivo</b>. <h3>Materials/Methods</h3> The modalities to research the effect of hypoxia <b>in vitro</b> includes clonogenic survival assay and γH2AX foci formation. Tumor growth delay with radiation and/or cisplatin, imaging study on spatial biodistribution between blood perfusion (Hoechst 33342), endogenous (eGFP and CA9) and exogenous (pimonidazole) hypoxia markers were used to investigate the effect of hypoxia <b>in vivo</b>. <h3>Results</h3> The constructed hypoxia-driven dual-reporter model contains 9 repeats of hypoxia responsive element and fusion gene of 9xHRE-HSV1-TKeGFP in NPC cells (abbreviated as SUNE-1/HRE cells) in which hypoxia-induced molecular events can be visualized by fluorescence or nuclear imaging techniques. There is no difference in radiosensitivity between SUNE1/Parental and SUNE1/HRE cells, while cisplatin significantly increases radiosensitivity in both cell lines. γH2AX serves as a surrogate for unrepaired DSB. When the cells are exposed to 0.5% O₂, eGFP is observed only in SUNE1/HRE cells. DSB repair rate under hypoxia is similar in SUNE1/Parental and SUNE1/HRE cells. The number of γH2AX foci is dramatically higher in the presence of cisplatin compared to that given irradiation alone under the condition of normoxia or hypoxia. It indicates that the addition of cisplatin enhances radiosensitivity by compromising the ability of cells to repair DSB. Cisplatin alone shows modest whereas irradiation moderate effect on delayed tumor growth. Combined treatment of irradiation and cisplatin further enhances the effect on tumor growth delay. High expression of eGFP is observed in the nonperfused regions. The spatial biodistribution of hypoxia-induced expression of eGFP reporter gene is similar to other endogenous or exogenous hypoxia biomarkers, i.e., CA9 and pimonidazole. <h3>Conclusion</h3> The build-in hypoxia-driven dual reporter 9HRE-HSV1-TKeGFP hypoxia research model does not modify intrinsic radiosensitivity of SUNE1 cells under the condition of normoxia or hypoxia, suggesting it be used as preclinical model to explore the therapeutic efficacy of various treatment modalities to overcome hypoxia by fluorescence or nuclear imaging techniques.

Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA

mRNA is a blooming technology for vaccination and has gained global attention during the SARS-CoV-2 pandemic. However, the recent clinical trials have highlighted increased reactogenicity when using high mRNA doses. Intending to increase the potency of mRNA therapeutics and to decrease the therapeutic dose, we designed a mRNA backbone and optimized the mRNA purification process. We used the enhanced green fluorescent protein (eGFP) reporter gene flanked by one 5′ untranslated region (UTR) and two 3′ UTRs of the human β-globin as a reference mRNA and identified the most promising mRNA sequence using in vitro and in vivo models. First, we assessed the impact of different poly(A) sizes on translation and selected the most optimal sequence. Then, we selected the best 5′ UTR among synthetic sequences displaying a high ribosome loading. Finally, we evaluated the transfection efficiency of our standard mRNA template after two capping strategies and purification using either double-stranded RNA (dsRNA) depletion or dephosphorylation of 5′PPP RNA or both combined. Double purification was shown to give the best results. Altogether, the use of a newly defined 5′ UTR coupled to post-transcriptional treatments will be of great interest in the mRNA vaccine field, by limiting the amount of the antigen-coding transcript and subsequently the formulation components needed for an efficient vaccination.

Expression of functional eGFP-fused antigen-binding fragment of ranibizumab in Pichia pastoris.

Introduction: Ranibizumab is a mouse monoclonal antibody fragment antigen-binding (Fab) against human vascular endothelial growth factor-A (VEGF-A), inhibiting angiogenesis. This antibody is commercially produced in Escherichia coli host and used to treat wet age-related macular degeneration (AMD). Methods: In this study, the heavy and light chains of ranibizumab were expressed in Pichia pastoris. The expressed chains were incubated overnight at 4°C for interaction. The formation of an active structure was evaluated based on the interaction with substrate VEGF-A using an indirect ELISA, and an electrochemical setup. Furthermore, reconstruction of split enhanced green fluorescent protein (eGFP) reporter, chimerized at the C-terminus of the heavy and light chains, was used to characterize chains’ interaction. Results: P. pastoris efficiently expressed designed constructs and secreted them into the culture medium. The anti-Fab antibody detected the constructed Fab structure in western blot analysis. Reconstruction of the split reporter confirmed the interaction between heavy and light chains. The designed ELISA and electrochemical setup results verified the binding activity of the recombinant Fab structure against VEGF-A. Conclusion: In this work, we indicated that the heavy and light chains of ranibizumab Fab fragments (with or without linkage to split parts of eGFP protein) were produced in P. pastoris. The fluorescence of reconstructed eGFP was detected after incubating the equal ratio of chimeric-heavy and light chains. Immunoassay and electrochemical tests verified the bioactivity of constructed Fab. The data suggested that P. pastoris could be considered a potential efficient eukaryotic host for ranibizumab production.

Sphingolipid extracts enhance gene delivery of cationic lipid vesicles into retina and brain

The aim was to evaluate relevant biophysic processes related to the physicochemical features and gene transfection mechanism when sphingolipids are incorporated into a cationic niosome formulation for non-viral gene delivery to central nervous system. For that, two formulations named niosphingosomes and niosomes devoid of sphingolipid extracts, as control, were developed by the oil-in water emulsion technique. Both formulations and the corresponding complexes, obtained upon the addition of the reporter EGFP plasmid, were physicochemically and biologically characterized and evaluated. Compared to niosomes, niosphingosomes, and the corresponding complexes decreased particle size and increased superficial charge. Although there were not significant differences in the cellular uptake, cell viability and transfection efficiency increased when human retinal pigment epithelial (ARPE-19) cells were exposed to niosphingoplexes. Endocytosis via caveolae decreased in the case of niosphingoplexes, which showed higher co-localization with lysosomal compartment, and endosomal escape properties. Moreover, niosphingoplexes transfected not only primary central nervous system cells, but also different cells in mouse retina, depending on the administration route, and brain cortex. These preliminary results suggest that niosphingosomes represent a promising non-viral vector formulation purposed for the treatment of both retinal and brain diseases by gene therapy approach.

The Dynamics of Axon Bifurcation Development in the Cerebral Cortex of Typical and Acallosal Mice

The corpus callosum (CC) is a major interhemispheric commissure of placental mammals. Early steps of CC formation rely on guidance strategies, such as axonal branching and collateralization. Here we analyze the time-course dynamics of axonal bifurcation during typical cortical development or in a CC dysgenesis mouse model. We use Swiss mice as a typical CC mouse model and find that axonal bifurcation rates rise in the cerebral cortex from embryonic day (E)17 and are reduced by postnatal day (P)9. Since callosal neurons populate deep and superficial cortical layers, we compare the axon bifurcation ratio between those neurons by electroporating ex vivo brains at E13 and E15, using eGFP reporter to label the newborn neurons on organotypic slices. Our results suggest that deep layer neurons bifurcate 32% more than superficial ones. To investigate axonal bifurcation in CC dysgenesis, we use BALB/c mice as a spontaneous CC dysgenesis model. BALB/c mice present a typical layer distribution of SATB2 callosal cells, despite the occurrence of callosal anomalies. However, using anterograde DiI tracing, we find that BALB/c mice display increased rates of axonal bifurcations during early and late cortical development in the medial frontal cortex. Midline guidepost cells adjacent to the medial frontal cortex are significant reduced in the CC dysgenesis mouse model. Altogether these data suggest that callosal collateral axonal exuberance is maintained in the absence of midline guidepost signaling and might facilitate aberrant connections in the CC dysgenesis mouse model.

Construction and Characterization of a Novel Bacmid AcBac-Syn Based on a Synthesized Baculovirus Genome

Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides. Homologous recombination and Bac-to-Bac system have been the main methods for manipulating the baculovirus genome. Recently, we generated a synthetic baculovirus AcMNPV-WIV-Syn1 which fully resembled its parental virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we report the modification of AcMNPV-WIV-Syn1 into a novel bacmid, AcBac-Syn, which can be used as a backbone for Bac-to-Bac system. To achieve this, a vector contained a LacZ:attTn7 and egfp cassette was constructed, and recombined with a linearized AcMNPV-WIV-Syn1 genome by transformation-associated recombination in yeast to generate bacmid AcBac-Syn. The bacmid was then transfected to insect cells and the rescued virus showed similar biological characteristics to the wild-type virus in terms of the kinetics of budded virus production, the morphology of occlusion bodies, and the oral infectivity in insect larvae. For demonstration, a red fluorescent protein gene Dsred was transposed into the attTn7 site by conventional Bac-to-Bac method, and the transfection and infection assays showed that AcBac-Syn can be readily used for foreign gene insertion and expression. AcBac-Syn has several advantages over the conventional AcMNPV bacmids, such as it contains an egfp reporter gene which facilitates visualization of virus propagation and titration; its DNA copy numbers could be induced to a higher level in E. coli; and the retaining of the native polyhedrin gene in the genome making it an attractive system for studying the functions of gene related to occlusion body assembly and oral infection.

Construction and characterization of EGFP reporter plasmid harboring putative human RAX promoter for in vitro monitoring of retinal progenitor cells identity

BackgroundIn retinal degenerative disease, progressive and debilitating conditions result in deterioration of retinal cells and visual loss. In human, retina lacks the inherent capacity for regeneration. Therefore, regeneration of retinal layer from human retinal progenitor cells (hRPCs) is a challenging task and restricted in vitro maintenance of hRPCs remains as the main hurdle. Retina and anterior neural fold homeobox gene (RAX) play critical roles in developing retina and maintenance of hRPCs. In this study, for the first time regulatory regions of human RAX gene with potential promoter activity were experimentally investigated.ResultsFor this purpose, after in silico analysis of regulatory regions of human RAX gene, the expression of EGFP reporter derived by putative promoter sequences was first evaluated in 293 T cells and then in hRPCS derived from human embryonic stem cells. The candidate region (RAX-3258 bp) showed the highest EGFP expression in hRPCs. This reporter construct can be used for in vitro monitoring of hRPC identity and verification of an efficient culture medium for maintenance of these cells.ConclusionsFurthermore, our findings provide a platform for better insight into regulatory regions of human RAX gene and molecular mechanisms underlying its vital functions in retina development.

Facile method for delivering chikungunya viral replicons into mosquitoes and mammalian cells

Reverse genetics is an important tool in the elucidation of viral replication and the development of countermeasures; however, these methods are impeded by laborious and inefficient replicon delivery methods. This paper demonstrates the use of a baculovirus to facilitate the efficient delivery of autonomous CHIKV replicons into mosquito and mammalian cells in vitro as well as adult mosquitoes in vivo. The efficacy of this approach was verified via co-localization among an eGFP reporter, nsP1, and dsRNA as well as through the inhibition of an RNA-dependent RNA polymerase (RdRp) null mutation (DDAA) in nsP4, or the treatment of a known antiviral compound (6-azauridine). We also investigated the correlation between CHIKV replicon-launched eGFP expression and the effectiveness of CHIKV replicon variants in inducing IFN-β expression in human cell lines. This delivery method based on a single vector is applicable to mosquito and mammalian cells in seeking to decipher the mechanisms underlying CHIKV replication, elucidate virus–host interactions, and develop antivirals. This study presents an effective alternative to overcome many of the technological issues related to the study and utilization of autonomous arbovirus replicons.

Functional in vivo characterization of sox10 enhancers in neural crest and melanoma development

The role of a neural crest developmental transcriptional program, which critically involves Sox10 upregulation, is a key conserved aspect of melanoma initiation in both humans and zebrafish, yet transcriptional regulation of sox10 expression is incompletely understood. Here we used ATAC-Seq analysis of multiple zebrafish melanoma tumors to identify recurrently open chromatin domains as putative melanoma-specific sox10 enhancers. Screening in vivo with EGFP reporter constructs revealed 9 of 11 putative sox10 enhancers with embryonic activity in zebrafish. Focusing on the most active enhancer region in melanoma, we identified a region 23 kilobases upstream of sox10, termed peak5, that drives EGFP reporter expression in a subset of neural crest cells, Kolmer-Agduhr neurons, and early melanoma patches and tumors with high specificity. A ~200 base pair region, conserved in Cyprinidae, within peak5 is required for transgenic reporter activity in neural crest and melanoma. This region contains dimeric SoxE/Sox10 dimeric binding sites essential for peak5 neural crest and melanoma activity. We show that deletion of the endogenous peak5 conserved genomic locus decreases embryonic sox10 expression and disrupts adult stripe patterning in our melanoma model background. Our work demonstrates the power of linking developmental and cancer models to better understand neural crest identity in melanoma.

A cleavage-based surrogate reporter for the evaluation of CRISPR-Cas9 cleavage efficiency.

CRISPR–Cas9 is a powerful tool for genome engineering, but its efficiency largely depends on guide RNA (gRNA). There are multiple methods available to evaluate the efficiency of gRNAs, including the T7E1 assay, surveyor nuclease assay, deep sequencing, and surrogate reporter systems. In the present study, we developed a cleavage-based surrogate that we have named the LacI-reporter to evaluate gRNA cleavage efficiency. The LacI repressor, under the control of the EF-1α promoter, represses luciferase or EGFP reporter expression by binding to the lac operator. Upon CRISPR–Cas9 cleavage at a target site located between the EF-1α promoter and the lacI gene, repressor expression is disrupted, thereby triggering luciferase or EGFP expression. Using this system, we can quantitate gRNA cleavage efficiency by assessing luciferase activity or EGFP expression. We found a strong positive correlation between the cleavage efficiency of gRNAs measured using this reporter and mutation frequency, measured using surveyor and deep sequencing. The genome-editing efficiency of gRNAs was validated in human liver organoids. Our LacI-reporter system provides a useful tool to select efficient gRNAs for genome editing.

Herbal Extract from Codonopsis pilosula (Franch.) Nannf. Enhances Cardiogenic Differentiation and Improves the Function of Infarcted Rat Hearts.

Background: The roots of Codonopsis pilosula (Franch.) Nannf. have been used in traditional Chinese medicine for treating cardiovascular disease. In the current study, we aimed to discover herbal extracts from C. pilosula that are capable of improving cardiac function of infarcted hearts to develop a potential therapeutic approach. Methods: A mouse embryonic stem (ES) cell-based model with an enhanced green fluorescent protein (eGFP) reporter driven by a cardiomyocyte-specific promoter, the α-myosin heavy chain, was constructed to evaluate the cardiogenic activity of herbal extracts. Then, herbal extracts from C. pilosula with cardiogenic activity based on an increase in eGFP expression during ES cell differentiation were further tested in a rat myocardial infarction model with left anterior descending artery (LAD) ligation. Cardiac function assessments were performed using echocardiography, 1, 3, and 6 weeks post LAD ligation. Results: The herbal extract 417W from C. pilosula was capable of enhancing cardiogenic differentiation in mouse ES cells in vitro. Echocardiography results in the LAD-ligated rat model revealed significant improvements in the infarcted hearts at least 6 weeks after 417W treatment that were determined based on left ventricle fractional shortening (FS), fractional area contraction (FAC), and ejection fraction (EF). Conclusions: The herbal extract 417W can enhance the cardiogenic differentiation of ES cells and improve the cardiac function of infarcted hearts.

An efficient vector-based CRISPR/Cas9 system in an Oreochromis mossambicus cell line using endogenous promoters

CRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.

Tmem100-BAC-EGFP mice to selectively mark and purify embryonic endothelial cells of large caliber arteries in mid-gestational vascular formation.

Embryonic vascular development is achieved through the complex arrays of differentiation, proliferation, migration and mutual interaction of different cell types, and visualization as well as purification of unique cell populations are fundamental in studying its detailed mechanisms using in vivo experimental models. We previously demonstrated that Tmem100 was a novel endothelial gene encoding a small transmembrane protein, and that Tmem100 null mice showed embryonic lethality due to severe impairment of vascular formation. In the present study, we generated an EGFP reporter mouse line using a 216 kb genomic region containing mouse Tmem100 gene. A novel line designated as Tmem100-BAC-EGFP mice precisely recapitulated the Tmem100 expression profile at the mid-gestational stage, which was highly enriched in endothelial cells of large caliber arteries in mouse embryos. FACS experiments demonstrated that Tmem100-BAC-EGFP mice served to selectively purify a specific population of arterial endothelial cells, indicating their usefulness not only for the research concerning Tmem100 expression and function but also for comparative analysis of multiple endothelial cell subgroups in embryonic vascular development.

The baculovirus promoter OpIE2 sequence has inhibitory effect on the activity of the cytomegalovirus (CMV) promoter in HeLa and HEK-293T cells.

Understanding how promoters work in non-host cells is complex. Nonetheless, understanding this process is crucial while performing gene expression modulation studies. This study began with the process of constructing a shuttle vector with CMV and OpIE2 promoters in a tandem arrangement to achieve gene expression in both mammalian and insect cells, respectively. In this system, inhibitory regions in the 5' end of the OpIE2 insect viral promoter were found to be blocking the activity of the CMV promoter in mammalian cells. Initially, the OpIE2 promoter was cloned downstream of the CMV promoter and upstream of the EGFP reporter gene. After introducing the constructed shuttle vector to insect and mammalian cells, a significant drop in the CMV promoter activity in mammalian cells was observed. To enhance the CMV promoter activity, several modifications were made to the shuttle vector including site-directed mutagenesis to remove all ATG codons from the downstream promoter (OpIE2), separating the two promoters to eliminate the effect of transcription interference between them, and finally, identifying some inhibitory regions in the OpIE2 promoter sequence. When these inhibitory regions were removed, high expression levels in insect and mammalian cells were maintained. In conclusion, a shuttle vector was constructed that works efficiently in both mammalian and insect cell lines in the absence of baculovirus infection or gene expression. Moreover, the shuttle vector can be used as a platform to further study the reason for this inhibition, which may give new insights about transcription and promoters' mode of action in both insect and mammalian hosts.

Development of epithelial cholinergic chemosensory cells of the urethra and trachea of mice

Cholinergic chemosensory cells (CCC) are infrequent epithelial cells with immunosensor function, positioned in mucosal epithelia preferentially near body entry sites in mammals including man. Given their adaptive capacity in response to infection and their role in combatting pathogens, we here addressed the time points of their initial emergence as well as their postnatal development from first exposure to environmental microbiota (i.e., birth) to adulthood in urethra and trachea, utilizing choline acetyltransferase (ChAT)-eGFP reporter mice, mice with genetic deletion of MyD88, toll-like receptor-2 (TLR2), TLR4, TLR2/TLR4, and germ-free mice. Appearance of CCC differs between the investigated organs. CCC of the trachea emerge during embryonic development at E18 and expand further after birth. Urethral CCC show gender diversity and appear first at P6-P10 in male and at P11-P20 in female mice. Urethrae and tracheae of MyD88- and TLR-deficient mice showed significantly fewer CCC in all four investigated deficient strains, with the effect being most prominent in the urethra. In germ-free mice, however, CCC numbers were not reduced, indicating that TLR2/4-MyD88 signaling, but not vita-PAMPs, governs CCC development. Collectively, our data show a marked postnatal expansion of CCC populations with distinct organ-specific features, including the relative impact of TLR2/4-MyD88 signaling. Strong dependency on this pathway (urethra) correlates with absence of CCC at birth and gender-specific initial development and expansion dynamics, whereas moderate dependency (trachea) coincides with presence of first CCC at E18 and sex-independent further development.

Fine gene expression regulation by minor sequence variations downstream of the polyadenylation signal.

The termination of transcription is a complex process that substantially contributes to gene regulation in eukaryotes. Previously, it was noted that a single cytosine deletion at the position + 32bp relative to the single polyadenylation signal AAUAAA (hereafter the dC mutation) causes a 2-fold increase in the transcription level of the upstream eGFP reporter in mouse embryonic stem cells. Here, we analyzed the conservation of this phenomenon in immortalized mouse, human and drosophila cell lines and the influence of the dC mutation on the choice of the pre-mRNA cleavage sites. We have constructed dual-reporter plasmids to accurately measure the effect of the dC and other nearby located mutations on eGFP mRNA level by RT-qPCR. In this way, we found that the dC mutation leads to a 2-fold increase in the expression level of the upstream eGFP reporter gene in cultured mouse and human, but not in drosophila cells. In addition, 3' RACE analysis demonstrated that eGFP pre-mRNAs are cut at multiple positions between + 14 to + 31, and that the most proximal cleavage site becomes almost exclusively utilized in the presence of the dC mutation. We alsoidentified new short sequence variations located within positions + 25.. + 40 and + 33.. + 48 that increase eGFP expression up to ~2-4-fold. Altogether, the positive effect of the dC mutation seems to be conserved in mouse embryonic stem cells, mouse embryonic 3T3 fibroblasts and human HEK293T cells. In the latter cells, the dC mutation appears to be involved in regulating pre-mRNA cleavage site selection. Finally, a multiplexed approach is proposed to identify motifs located downstream of cleavage site(s) that are essential for transcription termination.