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https://www.readbyqxmd.com/read/28809021/genetic-manipulation-of-the-avian-urogenital-system-using-in-ovo-electroporation
#1
Claire E Hirst, Olivier Serralbo, Katie L Ayers, Kelly N Roeszler, Craig A Smith
One of the advantages of the avian embryo as an experimental model is its in ovo development and hence accessibility for genetic manipulation. Electroporation has been used extensively in the past to study gene function in chicken and quail embryos . Readily accessible tissues such as the neural tube, somites, and limb bud, in particular, have been targeted. However, more inaccessible tissues, such as the embryonic urogenital system , have proven more challenging to study. Here, we describe the use of in ovo electroporation of TOL2 vectors or RCASBP avian viral vectors for the rapid functional analysis of genes involved in avian sex determination and urogenital development ...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28767277/using-zebrafish-to-test-the-genetic-basis-of-human-craniofacial-diseases
#2
R Grecco Machado, B Frank Eames
Genome-wide association studies (GWASs) opened an innovative and productive avenue to investigate the molecular basis of human craniofacial disease. However, GWASs identify candidate genes only; they do not prove that any particular one is the functional villain underlying disease or just an unlucky genomic bystander. Genetic manipulation of animal models is the best approach to reveal which genetic loci identified from human GWASs are functionally related to specific diseases. The purpose of this review is to discuss the potential of zebrafish to resolve which candidate genetic loci are mechanistic drivers of craniofacial diseases...
August 1, 2017: Journal of Dental Research
https://www.readbyqxmd.com/read/28701772/genetic-targeting-and-anatomical-registration-of-neuronal-populations-in-the-zebrafish-brain-with-a-new-set-of-bac-transgenic-tools
#3
Dominique Förster, Irene Arnold-Ammer, Eva Laurell, Alison J Barker, António M Fernandes, Karin Finger-Baier, Alessandro Filosa, Thomas O Helmbrecht, Yvonne Kölsch, Enrico Kühn, Estuardo Robles, Krasimir Slanchev, Tod R Thiele, Herwig Baier, Fumi Kubo
Genetic access to small, reproducible sets of neurons is key to an understanding of the functional wiring of the brain. Here we report the generation of a new Gal4- and Cre-driver resource for zebrafish neurobiology. Candidate genes, including cell type-specific transcription factors, neurotransmitter-synthesizing enzymes and neuropeptides, were selected according to their expression patterns in small and unique subsets of neurons from diverse brain regions. BAC recombineering, followed by Tol2 transgenesis, was used to generate driver lines that label neuronal populations in patterns that, to a large but variable extent, recapitulate the endogenous gene expression...
July 12, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28518092/isolation-of-the-side-population-in-myc-induced-t-cell-acute-lymphoblastic-leukemia-in-zebrafish
#4
Margaret M Pruitt, Wilfredo Marin, Michael R Waarts, Jill L O de Jong
Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown...
May 4, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28252665/chromatin-states-shape-insertion-profiles-of-the-piggybac-tol2-and-sleeping-beauty-transposons-and-murine-leukemia-virus
#5
Junko Yoshida, Keiko Akagi, Ryo Misawa, Chikara Kokubu, Junji Takeda, Kyoji Horie
DNA transposons and retroviruses are versatile tools in functional genomics and gene therapy. To facilitate their application, we conducted a genome-wide insertion site profiling of the piggyBac (PB), Tol2 and Sleeping Beauty (SB) transposons and the murine leukemia virus (MLV) in mouse embryonic stem cells (ESCs). PB and MLV preferred highly expressed genes, whereas Tol2 and SB preferred weakly expressed genes. However, correlations with DNase I hypersensitive sites were different for all vectors, indicating that chromatin accessibility is not the sole determinant...
March 2, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28188691/zebrafish-transgenic-constructs-label-specific-neurons-in-xenopus-laevis-spinal-cord-and-identify-frog-v0v-spinal-neurons
#6
José L Juárez-Morales, Reyna I Martinez-De Luna, Michael E Zuber, Alan Roberts, Katharine E Lewis
A correctly functioning spinal cord is crucial for locomotion and communication between body and brain but there are fundamental gaps in our knowledge of how spinal neuronal circuitry is established and functions. To understand the genetic program that regulates specification and functions of this circuitry, we need to connect neuronal molecular phenotypes with physiological analyses. Studies using Xenopus laevis tadpoles have increased our understanding of spinal cord neuronal physiology and function, particularly in locomotor circuitry...
September 2017: Developmental Neurobiology
https://www.readbyqxmd.com/read/27994173/transgenic-overexpression-of-baff-regulates-the-expression-of-immune-related-genes-in-zebrafish-danio-rerio
#7
Li Zhang, Chao Liu, Xin Zhou, Ying Xie, Libo Su, Qi Geng, Binghui Liu, Shufeng Liu
The B-cell activating factor (BAFF) is a member of tumour necrosis factor (TNF) superfamily that specifically regulates B lymphocyte proliferation and survival. Excess BAFF leads to overproduction of antibodies for secretion, anti-dsDNA antibodies and a lupus-like syndrome in mice. To investigate whether transgenic overexpression of the zebrafish BAFF leads to immunoglobulin changes and/or early maturing of the immune system, a Tol2-GFP-2A-BAFF/His recombinant plasmid was constructed by inserting a 2A peptide between the green fluorescent protein (GFP) and BAFF sequences...
December 2016: Journal of Genetics
https://www.readbyqxmd.com/read/27858360/transgenic-zebrafish
#8
Xiaowen Chen, Dafne Gays, Massimo M Santoro
The generation and use of transgenic animals carrying exogenous pieces of DNA stably integrated in their genome is a quite common practice in modern laboratories. Zebrafish have been increasingly used for transgenesis studies mainly due to easy egg accessibility and manipulation together with relatively short generation time. The zebrafish transgenic technology becomes very useful when coupled to continuous in vivo observation of the vertebrate embryonic vasculature. Here we describe the most common technique to generate zebrafish transgenic fish using the Tol2-based methodology and their applications to visualize vascular tissues or endothelial cells in vivo and for functional tumor angiogenesis studies...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27797132/a-light-colored-region-of-caudal-fin-a-niche-of-melanocyte-progenitors-in-crucian-carp-cyprinus-carpio-l
#9
Yaping Huang, Yurong Luo, Jinhui Liu, Saiyu Gui, Mei Wang, Wenbin Liu, Liangyue Peng, Yamei Xiao
Melanocyte stem cells are a population of immature cells which sustain the self-renewal and replenish the differentiated melanocytes. In this research, a light-colored region (LCR) is observed at the heel of caudal fin in juvenile crucian carp. By cutting off the caudal fin, the operated caudal fin can regenerate in accordance with the original pigment pattern from the retained LCR. As markers of stem cells, Oct4 and Sox2 have been found to be highly expressed in the LCR as well as Mitfa, a label of the melanoblasts...
January 2017: Cell Biology International
https://www.readbyqxmd.com/read/27631880/a-tol2-gateway-compatible-toolbox-for-the-study-of-the-nervous-system-and-neurodegenerative-disease
#10
Emily K Don, Isabel Formella, Andrew P Badrock, Thomas E Hall, Marco Morsch, Elinor Hortle, Alison Hogan, Sharron Chow, Serene S L Gwee, Jack J Stoddart, Garth Nicholson, Roger Chung, Nicholas J Cole
Currently there is a lack in fundamental understanding of disease progression of most neurodegenerative diseases, and, therefore, treatments and preventative measures are limited. Consequently, there is a great need for adaptable, yet robust model systems to both investigate elementary disease mechanisms and discover effective therapeutics. We have generated a Tol2 Gateway-compatible toolbox to study neurodegenerative disorders in zebrafish, which includes promoters for astrocytes, microglia and motor neurons, multiple fluorophores, and compatibility for the introduction of genes of interest or disease-linked genes...
February 2017: Zebrafish
https://www.readbyqxmd.com/read/27503354/gal4-driver-transgenic-zebrafish-powerful-tools-to-study-developmental-biology-organogenesis-and-neuroscience
#11
REVIEW
K Kawakami, K Asakawa, M Hibi, M Itoh, A Muto, H Wada
Targeted expression by the Gal4-UAS system is a powerful genetic method to analyze the functions of genes and cells in vivo. Although the Gal4-UAS system has been extensively used in genetic studies in Drosophila, it had not been applied to genetic studies in vertebrates until the mid-2000s. This was mainly due to the lack of an efficient transgenesis tool in model vertebrates, such as the P-transposable element of Drosophila, that can create hundreds or thousands of transgene insertions in different loci on the genome and thereby enables the generation of transgenic lines expressing Gal4 in various tissues and cells via enhancer trapping...
2016: Advances in Genetics
https://www.readbyqxmd.com/read/27443928/contemporary-zebrafish-transgenesis-with-tol2-and-application-for-cre-lox-recombination-experiments
#12
A Felker, C Mosimann
Spatiotemporal transgene regulation by transgenic DNA recombinases is a central tool for reverse genetics in multicellular organisms, with excellent applications for misexpression and lineage tracing experiments. One of the most widespread technologies for this purpose is Cre recombinase-controlled lox site recombination that is attracting increasing interest in the zebrafish field. Tol2-mediated zebrafish transgenesis provides a stable platform to integrate lox cassette transgenes, while the amenability of the zebrafish embryo to drug treatments makes the model an ideal candidate for tamoxifen-inducible CreERT2 experiments...
2016: Methods in Cell Biology
https://www.readbyqxmd.com/read/27443927/transcriptional-regulation-using-the-q-system-in-transgenic-zebrafish
#13
A Ghosh, M E Halpern
Methods to label cell populations selectively or to modify their gene expression are critical tools in the study of developmental or physiological processes in vivo. A variety of approaches have been applied to the zebrafish model, capitalizing on Tol2 transposition to generate transgenic lines with high efficiency. Here we describe the adoption of the Q system of Neurospora crassa, which includes the QF transcription factor and the upstream activating sequence (QUAS) to which it binds. These components function as a bipartite regulatory system similar to that of yeast Gal4/UAS, producing robust expression in transient assays of zebrafish embryos injected with plasmids and in stable transgenic lines...
2016: Methods in Cell Biology
https://www.readbyqxmd.com/read/27443919/tol2-mediated-transgenesis-gene-trapping-enhancer-trapping-and-gal4-uas-system
#14
K Kawakami, K Asakawa, A Muto, H Wada
The Tol2 element is an active transposon that was found from the genome of the Japanese medaka fish. Since the Tol2 transposition system is active in all vertebrate cells tested so far, it has been applied to germ line transgenesis in various model animals including fish, frog, chicken, and mouse, and to gene transfer in culture cells. In zebrafish, the Tol2 system consists of the transposase mRNA and a Tol2 transposon-donor plasmid, and is introduced into fertilized eggs by microinjection. Thus genomic integrations of the Tol2 construct are generated in the germ lineage and transmitted to the offspring very efficiently...
2016: Methods in Cell Biology
https://www.readbyqxmd.com/read/27349114/-cashmere-goat-bacterial-artificial-chromosome-recombination-and-cell-transfection-system
#15
Tian Huang, Zhongyang Cao, Yaohui Yang, Gengsheng Cao
The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination...
March 2016: Sheng Wu Gong Cheng Xue Bao, Chinese Journal of Biotechnology
https://www.readbyqxmd.com/read/27318202/agin-measuring-the-landscape-of-cpg-methylation-of-individual-repetitive-elements
#16
Yuta Suzuki, Jonas Korlach, Stephen W Turner, Tatsuya Tsukahara, Junko Taniguchi, Wei Qu, Kazuki Ichikawa, Jun Yoshimura, Hideaki Yurino, Yuji Takahashi, Jun Mitsui, Hiroyuki Ishiura, Shoji Tsuji, Hiroyuki Takeda, Shinichi Morishita
MOTIVATION: Determining the methylation state of regions with high copy numbers is challenging for second-generation sequencing, because the read length is insufficient to map reads uniquely, especially when repetitive regions are long and nearly identical to each other. Single-molecule real-time (SMRT) sequencing is a promising method for observing such regions, because it is not vulnerable to GC bias, it produces long read lengths, and its kinetic information is sensitive to DNA modifications...
October 1, 2016: Bioinformatics
https://www.readbyqxmd.com/read/27259919/tol2-mediated-delivery-of-mirnas-to-the-chicken-otocyst-using-plasmid-electroporation
#17
Michelle L Stoller, Donna M Fekete
The avian embryo has a well-documented history as a model system for the study of neurogenesis, morphogenesis, and cell fate specification. This includes studies of the chicken inner ear that employ in ovo electroporation, in conjunction with the Tol2 system, to yield robust long-term transgene expression. Capitalizing on the success of this delivery method, we describe a modified version of the Tol2 expression vector that readily accepts the insertion of a microRNA-encoding artificial intron. This offers a strategy to investigate the possible roles of different candidate microRNAs in ear development by overexpression...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27214565/microinjection-for-transgenesis-and-genome-editing-in-threespine-sticklebacks
#18
Priscilla A Erickson, Nicholas A Ellis, Craig T Miller
The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes...
May 13, 2016: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/27120052/development-of-a-fluorescent-transgenic-zebrafish-biosensor-for-sensing-aquatic-heavy-metal-pollution
#19
Nilambari Pawar, P Gireesh-Babu, Supriya Sabnis, Kiran Rasal, Renuka Murthy, S G S Zaidi, Sridhar Sivasubbu, Aparna Chaudhari
We report a transgenic zebrafish (Danio rerio) designed to respond to heavy metals using a metal-responsive promoter linked to a fluorescent reporter gene (DsRed2). The metallothionein MT-Ia1 promoter containing metal-responsive elements was derived from the Asian green mussel, Perna viridis. The promoter is known to be induced by a broad spectrum of heavy metals. The promoter-reporter cassette cloned into the Tol2 transposon vector was microinjected into zebrafish embryos that were then reared to maturity...
October 2016: Transgenic Research
https://www.readbyqxmd.com/read/27042235/active-recombinant-tol2-transposase-for-gene-transfer-and-gene-discovery-applications
#20
Jun Ni, Kirk J Wangensteen, David Nelsen, Darius Balciunas, Kimberly J Skuster, Mark D Urban, Stephen C Ekker
BACKGROUND: The revolutionary concept of "jumping genes" was conceived by McClintock in the late 1940s while studying the Activator/Dissociation (Ac/Ds) system in maize. Transposable elements (TEs) represent the most abundant component of many eukaryotic genomes. Mobile elements are a driving force of eukaryotic genome evolution. McClintock's Ac, the autonomous element of the Ac/Ds system, together with hobo from Drosophila and Tam3 from snapdragon define an ancient and diverse DNA transposon superfamily named hAT...
2016: Mobile DNA
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