scholarly journals Stable transgenesis in Astyanax mexicanus using the Tol2 transposase system

2019 ◽  
Author(s):  
Bethany A. Stahl ◽  
Robert Peuß ◽  
Brittnee McDole ◽  
Alexander Kenzior ◽  
James B. Jaggard ◽  
...  
Keyword(s):  
New Technologies ◽  
Genetic Model ◽  
Model Organism ◽  
Cell Types ◽  
Model Systems ◽  
Transgenic Zebrafish ◽  
Specific Cell ◽  
Astyanax Mexicanus ◽  
Fish Model ◽  
Transgenic Lines

AbstractAstyanax mexicanus is a well-established and widely used fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. The establishment of A. mexicanus as an emergent model organism for understanding the evolutionary basis of development and behavior has been accelerated by an increasing availability of genomic approaches to identify genotype-phenotype associations. Despite important progress in the deployment of new technologies, deep mechanistic insights into A. mexicanus evolution and development have been limited by a lack of transgenic lines commonly used in genetic model systems. Here, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficient Tol2 system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter fused to eGFP expressed eGFP ubiquitously throughout development in a surface population of Astyanax. To define specific cell-types, we injected fish with a Cntnap2-mCherry construct that labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish. The lines provide proof-of-principle for the application of Tol2 transgenic technology in A. mexicanus. Expansion on these initial transgenic lines will provide a platform to address broadly important problems in the quest to bridge the genotype to phenotype gap.

scholarly journals Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish

2011 ◽  
Vol 105 (05) ◽  
pp. 811-819 ◽  
Author(s):  
Caroline Gray ◽  
Catherine Loynes ◽  
Moira Whyte ◽  
David Crossman ◽  
Stephen Renshaw ◽  
...  
Keyword(s):  
Tissue Injury ◽  
Transgenic Fish ◽  
Artificial Chromosome ◽  
Cell Types ◽  
Intravital Imaging ◽  
Transgenic Zebrafish ◽  
Specific Cell ◽  
Post Injury ◽  
Simultaneous Imaging ◽  
Neutrophil Number

SummaryThe zebrafish is an outstanding model for intravital imaging of inflammation due to its optical clarity and the ability to express fluorescently labelled specific cell types by transgenesis. However, although several transgenic labelling myeloid cells exist, none allow distinction of macrophages from neutrophils. This prevents simultaneous imaging and examination of the individual contributions of these important leukocyte subtypes during inflammation. We therefore used Bacterial Artificial Chromosome (BAC) recombineering to generate a transgenic Tg(fms:GAL4.VP16)i186, in which expression of the hybrid transcription factor Gal4-VP16 is driven by the fms (CSF1R) promoter. This was then crossed to a second transgenic expressing a mCherry-nitroreductase fusion protein under the control of the Gal4 binding site (the UAS promoter), allowing intravital imaging of mCherry-labelled macrophages. Further crossing this compound transgenic with the neutrophil transgenic Tg(mpx:GFP)i114 allowed clear distinction between macrophages and neutrophils and simultaneous imaging of their recruitment and behaviour during inflammation. Compared with neutrophils, macrophages migrate significantly more slowly to an inflammatory stimulus. Neutrophil number at a site of tissue injury peaked around 6 hours post injury before resolving, while macrophage recruitment increased until at least 48 hours. We show that macrophages were effectively ablated by addition of the prodrug metronidazole, with no effect on neutrophil number. Crossing with Tg(Fli1:GFP)y1 transgenic fish enabled intravital imaging of macrophage interaction with endothelium for the first time, revealing that endothelial contact is associated with faster macrophage migration. Tg(fms:GAL4.VP16)i186 thus provides a powerful tool for intravital imaging and functional manipulation of macrophage behaviour during inflammation.

scholarly journals Hooked on zebrafish: insights into development and cancer of endocrine tissues

2011 ◽  
Vol 18 (5) ◽  
pp. R149-R164 ◽  
Author(s):  
Caitlin Bourque ◽  
Yariv Houvras
Keyword(s):  
Genetic Model ◽  
Cancer Genetics ◽  
Model Organism ◽  
Specific Cell ◽  
Vertebrate Development ◽  
Cell Lineages ◽  
Endocrine Organs ◽  
And Function ◽  
Thyroid Development ◽  
Endocrine Tissues

Zebrafish is emerging as a unique model organism for studying cancer genetics and biology. For several decades zebrafish have been used to study vertebrate development, where they have made important contributions to understanding the specification and differentiation programs in many tissues. Recently, zebrafish studies have led to important insights into thyroid development, and have been used to model endocrine cancer. Zebrafish possess a unique set of attributes that make them amenable to forward and reverse genetic approaches. Zebrafish embryos develop rapidly and can be used to study specific cell lineages or the effects of chemicals on pathways or tissue development. In this review, we highlight the structure and function of endocrine organs in zebrafish and outline the major achievements in modeling cancer. Our goal is to familiarize readers with the zebrafish as a genetic model system and propose opportunities for endocrine cancer research in zebrafish.

scholarly journals Towards A Genetic Model Organism: An Efficient Method For Stable Genetic Transformation of Eschscholzia Californica (Ranunculales)

2021 ◽  
Author(s):  
Dominik Lotz ◽  
Jafargholi Imani ◽  
Katrin Ehlers ◽  
Annette Becker
Keyword(s):  
Genetic Transformation ◽  
Genetic Model ◽  
Function Analysis ◽  
Model Organism ◽  
Protoplast Isolation ◽  
Model Systems ◽  
Eschscholzia Californica ◽  
Genetic Manipulations ◽  
California Poppy ◽  
Gene Function Analysis

Abstract California poppy (Eschscholzia californica) is a member of the Ranunculales, the sister order to all other eudicots and as such in a phylogenetically highly informative position. Ranunculales are known for their diverse floral morphologies and biosynthesis of many pharmaceutically relevant alkaloids. E. californica it is widely used as model system to study the conservation of flower developmental control genes. However, within the Ranunculales, options for stable genetic manipulations are rare and genetic model systems are thus difficult to establish. Here, we present a method for the efficient and stable genetic transformation via Agrobacterium tumefaciens-mediated transformation, somatic embryo induction, and regeneration of E. californica. Further, we provide a rapid method for protoplast isolation and transformation. This allows the study of gene functions in a single-cell and full plant context to enable gene function analysis and modification of alkaloid biosynthesis pathways by e.g. genome editing techniques providing important genetic resources for the genetic model organism E. californica.

scholarly journals Translational stem cell therapy: vascularized skin grafts in skin repair and regeneration

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Qian Hua Phua ◽  
Hua Alexander Han ◽  
Boon-Seng Soh
Keyword(s):  
Tissue Engineering ◽  
New Technologies ◽  
Rapid Development ◽  
Cell Types ◽  
Skin Tissue ◽  
Skin Substitute ◽  
Specific Cell ◽  
Skin Grafts ◽  
Cell Therapies ◽  
Micro Patterning

AbstractThe skin is made up of a plethora of cells arranged in multiple layers with complex and intricate vascular networks, creating a dynamic microenvironment of cells-to-matrix interactions. With limited donor sites, engineered skin substitute has been in high demand for many therapeutic purposes. Over the years, remarkable progress has occurred in the skin tissue-engineering field to develop skin grafts highly similar to native tissue. However, the major hurdle to successful engraftment is the incorporation of functional vasculature to provide essential nutrients and oxygen supply to the embedded cells. Limitations of traditional tissue engineering have driven the rapid development of vascularized skin tissue production, leading to new technologies such as 3D bioprinting, nano-fabrication and micro-patterning using hydrogel based-scaffold. In particular, the key hope to bioprinting would be the generation of interconnected functional vessels, coupled with the addition of specific cell types to mimic the biological and architectural complexity of the native skin environment. Additionally, stem cells have been gaining interest due to their highly regenerative potential and participation in wound healing. This review briefly summarizes the current cell therapies used in skin regeneration with a focus on the importance of vascularization and recent progress in 3D fabrication approaches to generate vascularized network in the skin tissue graft.

scholarly journals Transgenesis in the acoel Hofstenia miamia

2021 ◽  
Author(s):  
Lorenzo Ricci ◽  
Mansi Srivastava
Keyword(s):  
Cell Biology ◽  
Transgene Expression ◽  
Cell Types ◽  
Whole Body ◽  
Specific Cell ◽  
Embryonic Cells ◽  
Mechanistic Studies ◽  
Transgene Integration ◽  
Transgenic Lines ◽  
New Research

The acoel worm Hofstenia miamia, which can replace tissue lost to injury via differentiation of a population of stem cells, has emerged as a new research organism for studying regeneration. To enhance the depth of mechanistic studies in this system, we devised a protocol for microinjection into embryonic cells that resulted in stable transgene integration into the genome and generated animals with tissue-specific fluorescent transgene expression in epidermis, gut, and muscle. We demonstrate that transgenic Hofstenia are amenable to the isolation of specific cell types, detailed investigations of regeneration, tracking of photoconverted molecules, and live imaging. Further, our stable transgenic lines revealed new insights into the biology of Hofstenia, unprecedented details of cell morphology and the organization of muscle as a cellular scaffold for other tissues. Our work positions Hofstenia as a powerful system with unparalleled tools for mechanistic investigations of development, whole-body regeneration, and stem cell biology.

Abstract 187: c-Kit Biology Revealed by Two Transgenic Reporter Models.

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Natalie A Gude ◽  
Fareheh Firouzi ◽  
Kristine Nguyen ◽  
Christina Payne ◽  
Veronica Sacchi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Transgenic Mouse ◽  
Cardiac Myocyte ◽  
Biological Significance ◽  
Cell Types ◽  
Experimental Models ◽  
Model Systems ◽  
Transgenic Lines

Background: The biological significance of c-Kit as a marker of cardiac stem cells, and role(s) of c-Kit+ cells in myocardial development or in response to pathologic injury remain unresolved due to varied findings among investigators and experimental model systems. Alternative experimental models and approaches are needed to achieve a broader perspective of cardiac c-Kit biology that contextualizes discrepant published observations. Objectives: Tracking c-Kit expression using transgenesis overcomes limitations inherent to knock-in reporter models. Two novel, inducible transgenic c-Kit reporter models are presented in this study to further elaborate on myocardial c-Kit biology. Methods: A previously characterized mouse c-Kit promoter segment was engineered to generate a transgenic mouse in which rtTA transactivator is expressed in c-Kit+ cells (c-KitrtTA). c-KitrtTA crossed to Tet-Responsive-Element(TRE)-Histone2B-EGFP or TRE-Cre lines produces the CKH2B and CKCre double transgenic lines, which express doxycycline-inducible H2BEGFP or Cre proteins in c-Kit+ cells. The CKmTmG triple transgenic mouse, arising from CKCre crossed to the ROSAmTmG reporter line, utilizes doxycycline induced recombination to tag c-Kit+ cells irreversibly with membrane bound EGFP. Endogenous c-Kit and transgenic reporter expression was assessed in adult cardiac myocyte and nonmyocyte cells from these mice under resting and cellular stress conditions using immunohistochemistry and flow cytometry. Results: Coincidence of c-Kit and EGFP is observed in approximately 75% of freshly isolated nonmyocyte cells as detected by flow cytometry. A subpopulation of cardiomyocytes express H2BEGFP or mEGFP in the uninjured, doxycycline treated adult heart. H2BEGFP and c-Kit expression increase in myocytes in response to isoproterenol-induced pathologic stress in vivo and in vitro. Conclusion: These c-Kit transgenic reporter models provide sensitive, specific, inducible and persistent tracking of c-Kit promoter activation. Results presented here reveal an unexpected role for c-Kit expression in adult cardiomyocytes. Future studies will use both models to investigate c-Kit expression in all cell types during cardiac formation and repair.

scholarly journals Photoperiod-responsive changes in chromatin accessibility in phloem-companion and epidermis cells of Arabidopsis leaves

2021 ◽  
Author(s):  
Hao Tian ◽  
Yuru Li ◽  
Ce Wang ◽  
Xingwen Xu ◽  
Yajie Zhang ◽  
...  
Keyword(s):  
Cell Types ◽  
Chromatin Accessibility ◽  
Day Length ◽  
Specific Cell ◽  
Long Day ◽  
Cell Tissue ◽  
Companion Cells ◽  
Transgenic Lines ◽  
Dna Insertion ◽  
Accessible Chromatin

Abstract Photoperiod plays a key role in controlling the phase transition from vegetative to reproductive growth in flowering plants. Leaves are the major organs perceiving day-length signals, but how specific leaf cell-types respond to photoperiod remains unknown. We integrated photoperiod-responsive chromatin accessibility and transcriptome data in leaf epidermis and vascular companion cells of Arabidopsis thaliana by combining INTACT (Isolation of Nuclei Tagged in specific Cell/Tissue types) with ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) and RNA-sequencing. Despite a large overlap, vasculature and epidermis cells responded differently. Long-day predominantly induced accessible chromatin regions (ACRs); in the vasculature, more ACRs were induced and these were located at more distal gene regions, compared with the epidermis. Vascular ACRs induced by long day were highly enriched in binding sites for flowering-related transcription factors. Among the highly ranked genes (based on chromatin and expression signatures in the vasculature), we identified TREHALOSE-6-PHOSPHATASE SYNTHASE 9 (TPS9) as a flowering activator, as shown by the late flowering phenotypes of T-DNA insertion mutants and transgenic lines with phloem-specific knockdown of TPS9. Our cell-type-specific analysis sheds light on how the long-day photoperiod stimulus impacts chromatin accessibility in a tissue-specific manner to regulate plant development.

scholarly journals High-resolution mapping of cancer cell networks using co-functional interactions

2018 ◽  
Author(s):  
Evan A. Boyle ◽  
Jonathan K. Pritchard ◽  
William J. Greenleaf
Keyword(s):  
Cancer Cell ◽  
New Technologies ◽  
Protein Complexes ◽  
Cell Types ◽  
Genetic Interactions ◽  
Model Systems ◽  
Detection Approach ◽  
Crispr Screen ◽  
Resolution Mapping ◽  
Cell Networks

AbstractPowerful new technologies for perturbing genetic elements have expanded the study of genetic interactions in model systems ranging from yeast to human cell lines. However, technical artifacts can confound signal across genetic screens and limit the immense potential of parallel screening approaches. To address this problem, we devised a novel PCA-based method for eliminating these artifacts and bolstering sensitivity and specificity for detection of genetic interactions. Applying this strategy to a set of >300 whole genome CRISPR screens, we report ~1 million pairs of correlated “co-functional” genes that provide finer-scale information about cell compartments, biological pathways, and protein complexes than traditional gene sets. Lastly, we employed a gene community detection approach to implicate core genes for cancer growth and compress signal from functionally related genes in the same community into a single score. This work establishes new algorithms for probing cancer cell networks and motivates the acquisition of further CRISPR screen data across diverse genotypes and cell types to further resolve the complexity of cell signaling processes.

scholarly journals Model systems for regeneration:Arabidopsis

Development ◽  
2021 ◽  
Vol 148 (6) ◽  
Author(s):  
Mabel Maria Mathew ◽  
Kalika Prasad
Keyword(s):  
Arabidopsis Thaliana ◽  
Plant Regeneration ◽  
Cell Walls ◽  
Molecular Mechanisms ◽  
Molecular Genetic ◽  
Cell Types ◽  
Model Systems ◽  
Specific Cell ◽  
Multi Scale ◽  
Perfect Model

ABSTRACTPlants encompass unparalleled multi-scale regenerative potential. Despite lacking specialized cells that are recruited to injured sites, and despite their cells being encased in rigid cell walls, plants exhibit a variety of regenerative responses ranging from the regeneration of specific cell types, tissues and organs, to the rebuilding of an entire organism. Over the years, extensive studies on embryo, shoot and root development in the model plant species Arabidopsis thaliana have provided insights into the mechanisms underlying plant regeneration. These studies highlight how Arabidopsis, with its wide array of refined molecular, genetic and cell biological tools, provides a perfect model to interrogate the cellular and molecular mechanisms of reprogramming during regeneration.

scholarly journals Time-resolved expression analysis comparing two selective retinal cell ablation paradigms in zebrafish reveals shared and cell-specific regenerative regulatory networks

2020 ◽  
Author(s):  
Steven L. Walker ◽  
Guohua Wang ◽  
Kevin B. Emmerich ◽  
Fang Wang ◽  
David T. White ◽  
...  
Keyword(s):  
Stem Cells ◽  
Regulatory Networks ◽  
Model Organism ◽  
Cell Loss ◽  
Cell Types ◽  
Retinal Cell ◽  
Specific Cell ◽  
Retinal Regeneration ◽  
Cell Degeneration ◽  
Specific Expression

AbstractZebrafish are an effective model organism for retinal regeneration studies. Regenerated retinal cells are derived from Müller glia (MG) stem cells. Mammalian MG can also produce new retinal neurons; however, this regenerative potential remains dormant in the absence of genetic and/or chemical stimulation. An understanding of how the regenerative potential of MG is regulated could aid efforts to promote regeneration therapeutically. Following widespread retinal cell death, developmental signaling coordinates regeneration. Less is known about how MG respond to the loss of specific cell types, i.e., paradigms similar to retinal degenerative diseases. To address this, transcriptomic responses to the selective loss of rod photoreceptors or retinal bipolar cells were compared over twelve timepoints spanning cell degeneration and regeneration. Shared and paradigm-specific expression changes were identified throughout regeneration. Overall, paradigm-specific changes predominated, suggesting cell-specific mechanisms for activating MG stem cells. One particularly interested finding new for retinal regeneration was early regulation of SOCS family genes. These are associated with stat3 activation and the JAK/STAT pathway which has been well documented in similar studies and was further supported in our analyses. These data support the concept that selective retinal cell loss can elicit cell-specific regenerative programs and provide novel insights into retinal regeneration.

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