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
Source: PubMed


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.

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    • "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
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    • "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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