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Nppa is a cardiac hormone which plays critical roles in regulating salt-water balance. Its expression is restricted to the atria of the healthy post-natal heart. During heart development, spatio-temporal expression of Nppa is dynamically changed within the heart and becomes restricted to the atria upon birth. In contrast to its atrial specific expression after birth, Nppa is re-expressed in the adult ventricles in response to cardiac hypertrophy. To study cardiac chamber specification during development and pathological cardiac remodeling during heart disease, we generated a novel Nppa reporter mouse line by knocking-in a tagBFP reporter cassette into 3'-UTR of the Nppa gene without disrupting the endogenous gene. Our results demonstrated dynamic tagBFP expression in the developing heart, recapitulating the spatiotemporal expression pattern of endogenous Nppa. We also found that Nppa-tagBFP is induced in the ventricle during pathological remodeling. Taken together, Nppa-tagBFP reporter knock-in mouse model described in this article will serve as a valuable tool to study cardiac chamber specification during development as well as pathological cardiac remodeling.
© 2019 Wiley Periodicals, Inc.
Flow cytometry studies on injured kidney tubules are complicated by the low yield of nucleated single cells. Furthermore, cell-specific responses such as cell cycle dynamics in vivo have conventionally relied on indirect immunohistochemistry and proximal tubule markers that may be downregulated in injury. Here, we report a new tissue dissociation protocol for the kidney with an early fixation step that greatly enhances the yield of single cells. Genetic labeling of the proximal tubule with either mT/mG "tomato" or R26Fucci2aR (Fucci) cell cycle reporter mice allows us to follow proximal tubule-specific changes in cell cycle after renal injury. Image-based flow cytometry (FlowSight) enables gating of the cell cycle and concurrent visualization of the cells with bright field and fluorescence. We used the Fucci mouse in conjunction with FlowSight to identify a discrete polyploid population in proximal tubules after aristolochic acid injury. The tissue dissociation protocol in conjunction with genetic labeling and image-based flow cytometry is a tool that can improve our understanding of any discrete cell population after injury.
Fluorescent protein reporter genes are widely used to identify and sort murine pancreatic β-cells. In this study, we compared use of the MIP-GFP transgene, which exhibits aberrant expression of human growth hormone (hGH), with a newly derived Ins2 allele that lacks hGH expression on the expression of sex-specific genes. β-Cells from MIP-GFP transgenic mice exhibit changes in the expression of 7,733 genes, or greater than half of their transcriptome, compared with β-cells from Ins2 mice. To determine how these differences might affect a typical differential gene expression study, we analyzed the effect of sex on gene expression using both reporter lines. Six hundred fifty-seven differentially expressed genes were identified between male and female β-cells containing the Ins2 allele. Female β-cells exhibit higher expression of Xist, Tmed9, Arpc3, Eml2, and several islet-enriched transcription factors, including Nkx2-2 and Hnf4a, whereas male β-cells exhibited a generally higher expression of genes involved in cell cycle regulation. In marked contrast, the same male vs. female comparison of β-cells containing the MIP-GFP transgene revealed only 115 differentially expressed genes, and comparison of the 2 lists of differentially expressed genes revealed only 17 that were common to both analyses. These results indicate that 1) male and female β-cells differ in their expression of key transcription factors and cell cycle regulators and 2) the MIP-GFP transgene may attenuate sex-specific differences that distinguish male and female β-cells, thereby impairing the identification of sex-specific variations.
MLC-2v is a myosin light chain regulatory protein which is specifically expressed in ventricular cardiomyocytes and slow twitch skeletal muscle cells. MLC-2v plays critical roles in ventricular maturation during heart development. Mice lacking MLC-2v are embryonic lethal due to heart failure associated with abnormal myofibrillar organization of ventricular cardiomyocytes. To study the development of ventricular cardiac muscle and slow twitch skeletal muscle, we generated a new MLC-2v reporter mouse line by knocking-in a tdTomato reporter cassette into 3' UTR of the MLC-2v gene without disrupting the endogenous gene. Our results demonstrated specific MLC-2v-tdTomato knock-in reporter expression in ventricular cardiomyocytes and slow twitch muscle during myogenesis, precisely recapitulating the spatiotemporal expression pattern of endogenous MLC-2v. No tdTomato expression was observed in the atria, fast twitch muscle or other organs throughout development into adulthood. Isolated neonatal and adult ventricular cardiomyocytes uniformly express tdTomato. Taken together, MLC-2v-tdTomato knock-in reporter mouse model described in this article will serve as a valuable tool to study cardiac chamber and skeletal muscle specification during development and regeneration by overcoming the pitfalls of transgenic strategies.
© 2018 Wiley Periodicals, Inc.
We used the CRISPR/Cas9 system to knock-in reporter transgenes at the kidney injury molecule-1 (KIM-1) locus and isolated human proximal tubule cell (HK-2) clones. PCR verified targeted knock-in of the luciferase and eGFP reporter at the KIM-1 locus. HK-2-KIM-1 reporter cells responded to various stimuli including hypoxia, cisplatin, and high glucose, indicative of upregulation of KIM-1 expression. We attempted using CRISPR/Cas9 to also engineer the KIM-1 reporter in telomerase-immortalized human RPTEC cells. However, these cells demonstrated an inability to undergo homologous recombination at the target locus. KIM-1-reporter human proximal tubular cells could be valuable tools in drug discovery for molecules inhibiting kidney injury. Additionally, our gene targeting strategy could be used in other cell lines to evaluate the biology of KIM-1 in vitro or in vivo.
While innate behaviors are conserved throughout the animal kingdom, it is unknown whether common signaling pathways regulate the development of neuronal populations mediating these behaviors in diverse organisms. Here, we demonstrate that the Wnt/ß-catenin effector Lef1 is required for the differentiation of anxiolytic hypothalamic neurons in zebrafish and mice, although the identity of Lef1-dependent genes and neurons differ between these 2 species. We further show that zebrafish and Drosophila have common Lef1-dependent gene expression in their respective neuroendocrine organs, consistent with a conserved pathway that has diverged in the mouse. Finally, orthologs of Lef1-dependent genes from both zebrafish and mouse show highly correlated hypothalamic expression in marmosets and humans, suggesting co-regulation of 2 parallel anxiolytic pathways in primates. These findings demonstrate that during evolution, a transcription factor can act through multiple mechanisms to generate a common behavioral output, and that Lef1 regulates circuit development that is fundamentally important for mediating anxiety in a wide variety of animal species.
Antibodies are the principal immune effectors that mediate protection against reinfection following viral infection or vaccination. Robust techniques for human mAb isolation have been developed in the last decade. The study of human mAbs isolated from subjects with prior immunity has become a mainstay for rational structure-based, next-generation vaccine development. The plethora of detailed molecular and genetic studies coupling the structure of antigen-antibody complexes with their antiviral function has begun to reveal common principles of critical interactions on which we can build better vaccines and therapeutic antibodies. This review outlines the approaches to isolating and studying human antiviral mAbs and discusses the common principles underlying the basis for their activity. This review also examines progress toward the goal of achieving a comprehensive understanding of the chemical and physical basis for molecular recognition of viral surface proteins in order to build predictive molecular models that can be used for vaccine design.
Copyright © 2017. Published by Elsevier Inc.
EGF receptor (EGFR) is a critical signaling node throughout life. However, it has not been possible to directly visualize endogenous Egfr in mice. Using CRISPR/Cas9 genome editing, we appended a fluorescent reporter to the C terminus of the Egfr. Homozygous reporter mice appear normal and EGFR signaling is intact in vitro and in vivo. We detect distinct patterns of Egfr expression in progenitor and differentiated compartments in embryonic and adult mice. Systemic delivery of EGF or amphiregulin results in markedly different patterns of Egfr internalization and trafficking in hepatocytes. In the normal intestine, Egfr localizes to the crypt rather than villus compartment, expression is higher in adjacent epithelium than in intestinal tumors, and following colonic injury expression appears in distinct cell populations in the stroma. This reporter, under control of its endogenous regulatory elements, enables in vivo monitoring of the dynamics of Egfr localization and trafficking in normal and disease states.
Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Methods enabling kidney-specific gene transfer in adult mice are needed to develop new therapies for kidney disease. We attempted kidney-specific gene transfer following hydrodynamic tail vein injection using the kidney-specific podocin and gamma-glutamyl transferase promoters, but found expression primarily in the liver. In order to achieve kidney-specific transgene expression, we tested direct hydrodynamic injection of a DNA solution into the renal pelvis and found that luciferase expression was strong in the kidney and absent from extra-renal tissues. We observed heterogeneous, low-level transfection of the collecting duct, proximal tubule, distal tubule, interstitial cells, and rarely glomerular cells following injection. To assess renal injury, we performed the renal pelvis injections on uninephrectomised mice and found that their blood urea nitrogen was elevated at two days post-transfer but resolved within two weeks. Although luciferase expression quickly decreased following renal pelvis injection, the use of the piggyBac transposon system improved long-term expression. Immunosuppression with cyclophosphamide stabilised luciferase expression, suggesting immune clearance of the transfected cells occurs in immunocompetent animals. Injection of a transposon expressing erythropoietin raised the haematocrit, indicating that the developed injection technique can elicit a biologic effect in vivo. Hydrodynamic renal pelvis injection enables transposon mediated-kidney specific gene transfer in adult mice.