Poshusta, Tanya L.

Background: The extent to which tissue factor pathway inhibitor (TFPI) may regulate the extrinsic clotting cascade, atherogenesis, and MI is uncertain. Reverse genetic studies in the mouse, including deletion of the TFPI Kunitz-1 (K1) domain, have been hindered by intrauterine lethality. We sought to determine the role of TFPI in an alternative animal model, by knocking out the zebrafish ortholog tfpia . Methods and results: High-efficiency transcription activator-like effector nucleases (TALENs), targeting the 5’ region of tfpia, were microinjected into 1-cell zebrafish embryos. Sequence analysis of genomic DNA derived from adult caudal fin tissue revealed indels causing a frameshift mutation and premature stop codon prior to K1. Somatic mutants were outcrossed to WT fish, and the embryos were screened for germline mutations. Founders (F 1 ) were raised and incrossed to produce embryos (F 2 ), including approximately 25% with the desired biallelic null mutation ( tfpia -/- ) that were physically and developmentally indistinguishable, but functionally disparate from heterozygous ( tfpia +/- ) or WT ( tfpia +/+ ) siblings. At 6 days post-fertilization (dpf), F 2 tfpia -/- larvae exhibited a reduction in time to occlusion of the dorsal aorta after exposure to ferric chloride, which was more significant in 6-dpf F 3 tfpia -/- larvae (generated by incrossing F 2 tfpia -/- adults) lacking a possible maternal effect. Furthermore, F 2 tfpia -/- fish had in vivo evidence of increased fatty streaking as late larvae, after receiving a high cholesterol diet, and enhanced cardiac injury as adults, 4 days after cryoinjury-induced MI. Conclusions: Deletion of tfpia does not significantly effect zebrafish development, but leads to hypercoagulability in early larvae, enhanced atherogenesis in late larvae, and increased injury related to MI in adults, indicating a fundamental role for TFPI in the pathogenesis of cardiovascular disease.

Transcription activator-like effectors (TALEs) are extremely effective, single-molecule DNA-targeting molecular cursors used for locus-specific genome science applications, including high-precision molecular medicine and other genome engineering applications. TALEs are used in genome engineering for locus-specific DNA editing and imaging, as artificial transcriptional activators and repressors, and for targeted epigenetic modification. TALEs as nucleases (TALENs) are effective editing tools and offer high binding specificity and fewer sequence constraints toward the targeted genome than other custom nuclease systems. One bottleneck of broader TALE use is reagent accessibility. For example, one commonly deployed method uses a multitube, 5-day assembly protocol. Here we describe FusX, a streamlined Golden Gate TALE assembly system that (1) is backward compatible with popular TALE backbones, (2) is functionalized as a single-tube 3-day TALE assembly process, (3) requires only commonly used basic molecular biology reagents, and (4) is cost-effective. More than 100 TALEN pairs have been successfully assembled using FusX, and 27 pairs were quantitatively tested in zebrafish, with each showing high somatic and germline activity. Furthermore, this assembly system is flexible and is compatible with standard molecular biology laboratory tools, but can be scaled with automated laboratory support. To demonstrate, we use a highly accessible and commercially available liquid-handling robot to rapidly and accurately assemble TALEs using the FusX TALE toolkit. Together, the FusX system accelerates TALE-based genomic science applications from basic science screening work for functional genomics testing and molecular medicine applications.

AbstractGene regulation resulting from glucocorticoid receptor and glucocorticoid response element interactions is a hallmark feature of stress response signaling. Imbalanced glucocorticoid production and glucocorticoid receptor activity have been linked to socioeconomically crippling neuropsychiatric disorders, and accordingly there is a need to developin vivomodels to help understand disease progression and management. Therefore, we developed the transgenicSR4Gzebrafish reporter line with six glucocorticoid response elements used to promote expression of a short half‐life green fluorescent protein following glucocorticoid receptor activation. Herein, we document the ability of this reporter line to respond to both chronic and acute exogenous glucocorticoid treatment. The green fluorescent protein expression in response to transgene activation was high in a variety of tissues including the brain, and provided single‐cell resolution in the effected regions. The specificity of these responses is demonstrated using the partial agonist mifepristone and mutation of the glucocorticoid receptor. Importantly, the reporter line also modeled the temporal dynamics of endogenous stress response signaling, including the increased production of the glucocorticoid cortisol following hyperosmotic stress and the fluctuations of basal cortisol concentrations with the circadian rhythm. Taken together, these results characterize our newly developed reporter line for elucidating environmental or genetic modifiers of stress response signaling, which may provide insights to the neuronal mechanisms underlying neuropsychiatric disorders such as major depressive disorder.

Light chain (AL) amyloidosis is a devastating disease characterized by amyloid deposits formed by immunoglobulin light chains. Current available treatments involve conventional chemotherapy and autologous stem cell transplant. We have recently concluded a phase III trial comparing these two treatments. AL amyloidosis patients who achieve hematological complete response (CR) do not necessarily achieve organ response regardless of the treatment they received. In order to investigate the possible correlation between amyloid formation kinetics and organ response, we selected AL amyloidosis patients from the trial with kidney involvement and CR after treatment. Six patients were selected and their monoclonal immunoglobulin light chains were characterized. The proteins showed differences in their stability and their kinetics of amyloid formation. A correlation was detected at pH 7.4, showing that less stable proteins are more likely to form amyloid fibrils. AL-T03 is too unstable to form amyloid fibrils at pH 7.4. This protein was found in the only patient in the study that had organ response, suggesting that partially folded species are required for amyloid formation to occur in AL amyloidosis.