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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
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...
February 11, 2017: Developmental Neurobiology
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Giorgia Bergamin, Domenico Cieri, Giovanni Vazza, Francesco Argenton, Maria Luisa Mostacciuolo
OBJECTIVES: Deregulation of axonal transport in neurons is emerging as the major cause of many neurodegenerative diseases in human, such as Charcot-Marie-Tooth (CMT) neuropathy. However, little is known about how mitochondria move in vivo and whether cell culture systems truly represent what happens in living animals. Here we describe the generation of a new zebrafish transgenic line that specifically allows to study mitochondrial dynamics in motor neurons and its application to analyse mitochondrial movement in zebrafish models expressing CMT2A causing mutations...
June 2016: Biochimica et Biophysica Acta
Nozomu Takata, Eriko Sakakura, Takeya Kasukawa, Tetsushi Sakuma, Takashi Yamamoto, Yoshiki Sasai
The epiblast (foremost embryonic ectoderm) generates all three germ layers and therefore has crucial roles in the formation of all mammalian body cells. However, regulation of epiblast gene expression is poorly understood because of the difficulty of manipulating epiblast tissues in vivo. In the present study, using the self-organizing properties of embryonic stem cell (ESC), we generated and characterized epiblast-like tissue in three-dimensional culture. We identified significant genome-wide gene expression changes in this epiblast-like tissue by transcriptomic analysis...
June 2016: Human Gene Therapy
Pavle Vrljicak, Shijie Tao, Gaurav K Varshney, Helen Ngoc Bao Quach, Adita Joshi, Matthew C LaFave, Shawn M Burgess, Karuna Sampath
DNA transposons and retroviruses are important transgenic tools for genome engineering. An important consideration affecting the choice of transgenic vector is their insertion site preferences. Previous large-scale analyses of Ds transposon integration sites in plants were done on the basis of reporter gene expression or germ-line transmission, making it difficult to discern vertebrate integration preferences. Here, we compare over 1300 Ds transposon integration sites in zebrafish with Tol2 transposon and retroviral integration sites...
2016: G3: Genes—Genomes—Genetics
Fernando Fuentes, Eric Reynolds, Stephen W Lewellis, Gayatri Venkiteswaran, Holger Knaut
Transgenesis of large DNA constructs is essential for gene function analysis. Recently, Tol2 transposase-mediated transgenesis has emerged as a powerful tool to insert bacterial artificial chromosome (BAC) DNA constructs into the genome of zebrafish. For efficient transgenesis, the genomic DNA piece in the BAC construct needs to be flanked by Tol2 transposon sites, and the constructs should contain a transgenesis marker for easy identification of transgenic animals. We report a set of plasmids that contain targeting cassettes that allow the insertion of Tol2 sites and different transgenesis markers into BACs...
2016: G3: Genes—Genomes—Genetics
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