Generating mutant rats using the Sleeping Beauty transposon system
Laboratory of Mammalian Genetics, Division of Genome Dynamics, Creative Research Initiative "Sousei", Hokkaido University, North 10 West 8, Kita-ku, Sapporo 060-0810, Japan. Methods
(Impact Factor: 3.65).
05/2009; 49(3):236-42. DOI: 10.1016/j.ymeth.2009.04.010
The laboratory rat is an invaluable animal model for biomedical research. However, mutant rat resource is still limited, and development of methods for large-scale generation of mutants is anticipated. We recently utilized the Sleeping Beauty (SB) transposon system to develop a rapid method for generating insertional mutant rats. Firstly, transgenic rats carrying single transgenes, namely the SB transposon vector and SB transposase, were generated. Secondly, these single transgenic rats were interbred to obtain doubly-transgenic rats carrying both transgenes. The SB transposon was mobilized in the germline of these doubly-transgenic rats, reinserted into another location in the genome and heterozygous mutant rats were obtained in the progeny. Gene insertion events were rapidly and non-invasively identified by the green fluorescence protein (GFP) reporter incorporated in the transposon vector, which utilizes a polyA-trap approach. Mutated genes were confirmed by either linker ligation-mediated PCR or 3'-rapid amplification of cDNA ends (3'RACE). Endogenous expression profile of the mutated gene can also be visualized using the LacZ gene incorporated as a promoter-trap unit in the transposon vector. This method is straightforward, readily applicable to other transposon systems, and will be a valuable mutant rat resource to the biomedical research community.
Available from: EN-Min Zhou
- "The Sleeping Beauty (SB) is the first DNA transposon system that has been described to work in mammalian cells and is also the most widely used transposon system for mammalian transgenesis and mutagenesis (Takeda et al., 2007; Wang et al., 2008). For example , SB has been used to identify new cancer genes (Collier et al., 2005; Dupuy et al., 2006) and generate transgene animals (Kitada et al., 2009). However, SB transposition application is limited by its relatively low transposition efficiency and its nonspecific integration (Wu et al., 2006; Yant et al., 2005). "
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ABSTRACT: The PiggyBac (PB) transposon system is a non-viral DNA-transfer system in which a transposase directs integration of a PB transposon into a TTAA site in the genome. Transgenic expression of porcine CD163 is necessary and sufficient to confer non-permissive cells susceptible to infection with porcine reproductive and respiratory syndrome virus (PRRSV). Such permissive cells can be used as a tool for PRRSV cellular receptor and other studies. One of the problems in studying PRRSV is the lack of porcine cell lines. In this study, efficient transfection and expression of porcine CD163 in PK-15 cells by PB transposition was demonstrated. The stable PK-15(CD163) cell line was used in PRRSV infection assays. The data indicated that the average PB transgene copy number per genome was approximately ten. In line with previous literature the integration of PB into the genome had a bias toward the TTAA chromosomal site. The PK-15(CD163) cell line was susceptible to infection by different PRRSV strains and the virus grew to similar titers compared to the Marc-145 cell line. This simplification of PK-15(CD163) cell line production will provide a valuable tool to facilitate PRRSV cellular receptor studies and to accelerate existing vectors for PK-15 cell-based gene transfer and expression.
Journal of virological methods 07/2013; 193(2). DOI:10.1016/j.jviromet.2013.06.035 · 1.78 Impact Factor
Available from: Hans Hedrich
- "Aside from the pronuclear injection of recombinant DNA constructs, the integration of lentiviral vectors into the rat genome after injection into the perivitelline space and transposon-mediated insertions have enabled studies in transgenic rats [1-5]. Random integration of Sleeping Beauty and PiggyBac transposons caused gene inactivation at the integration sites [6,7]. Moreover, the cultivation of rat embryonic stem cells does allow gene targeting and functional deletion through homologous recombination (HR) in vitro[8-11]. "
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Engineered zinc-finger nucleases (ZFN) represented an innovative method for the genome manipulation in vertebrates. ZFN introduced targeted DNA double strand breaks (DSB) and initiated non-homologous end joining (NHEJ) after pronuclear or cytoplasmatic microinjection into zygotes. Resulting frame shift mutations led to functional gene ablations in zebra fish, mice, pigs and also in laboratory rats. Therefore, we targeted the rat Rag1 gene essential for the V(D)J recombination within the immunoglobulin production process and for the differentiation of mature B and T lymphocytes to generate an immunodeficient rat model in the LEW/Ztm strain.
After microinjection of Rag1 specific ZFN mRNAs in 623 zygotes of inbred LEW/Ztm rats 59 offspring were born from which one carried a 4 bp deletion. This frame shift mutation led to a premature stop codon and a subsequently truncated Rag1 protein confirmed by the loss of the full-length protein in Western Blot analysis. Truncation of the Rag1 protein was characterized by the complete depletion of mature B cells. The remaining T cell population contained mature CD4+/CD3+/TCRαβ+ as well as CD8+/CD3+/TCRαβ+ positive lymphocytes accompanied by a compensatory increase of natural killer cells in the peripheral blood. Reduction of T cell development in Rag1 mutant rats was associated with a hypoplastic thymus that lacked follicular structures. Histological evaluation also revealed the near-complete absence of lymphocytes in spleen and lymph nodes in the immunodeficient Rag1 mutant rat.
The Rag1 mutant rat will serve as an important model for transplantation studies. Furthermore, it may be used as a model for reconstitution experiments related to the immune system, particularly with respect to different populations of human lymphocytes, natural killer cells and autoimmune phenomena.
BMC Immunology 11/2012; 13(1):60. DOI:10.1186/1471-2172-13-60 · 2.48 Impact Factor
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ABSTRACT: This article summarizes the proceedings of a one-day international workshop held in July 2009 on the role of HLA-B27 in the pathogenesis of ankylosing spondylitis (AS) and related disorders. HLA-B27 is found in about 90% of patients with AS, with an odds ratio of about 100, but the mechanism underlying this association is not known. There are currently 3 major mechanistic hypotheses for this association: (1) T cell recognition of one or more B27 presented peptides; (2) B27 heavy-chain misfolding that induces an unfolded protein response; and (3) innate immune recognition of cell-surface expressed B27 heavy-chain dimers. None of these hypotheses accounts for the tissue specificity of the inflammation characteristic of AS. These hypotheses were discussed in the context of known epidemiologic, biochemical, structural, and immunologic differences among HLA-B27 subtypes; data from the HLA-B27 transgenic rat model of spondyloarthritis; the growing list of other genes that have been found to be associated with AS; and other data on the pathogenesis of spondyloarthritis. Proposed directions for future research include expanded efforts to define similarities and differences among the B27 subtypes; further development of animal models; identifying the interactions of B27 with the products of other genes associated with AS; and continued investigation into the pathogenesis of spondyloarthritis.
The Journal of Rheumatology 12/2010; 37(12):2606-16. DOI:10.3899/jrheum.100889 · 3.19 Impact Factor
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