Efficient mouse transgenesis using Gateway-compatible ROSA26 locus targeting vectors and F1 hybrid ES cells

Department for Molecular Biomedical Research, VIB, Gent, Belgium.
Nucleic Acids Research (Impact Factor: 9.11). 04/2009; 37(7):e55. DOI: 10.1093/nar/gkp112
Source: PubMed


The ability to rapidly and efficiently generate reliable Cre/loxP conditional transgenic mice would greatly complement global high-throughput gene targeting initiatives aimed at identifying gene function in the mouse. We report here the generation of Cre/loxP conditional ROSA26-targeted ES cells within 3-4 weeks by using Gateway cloning to build the target vectors. The cDNA of the gene of interest can be expressed either directly by the ROSA26 promoter providing a moderate level of expression or by a CAGG promoter placed in the ROSA26 locus providing higher transgene expression. Utilization of F1 hybrid ES cells with exceptional developmental potential allows the production of germ line transmitting, fully or highly ES cell-derived mice by aggregation of cells with diploid embryos. The presented streamlined procedures accelerate the examination of phenotypical consequences of transgene expression. It also provides a unique tool for comparing the biological activity of polymorphic or splice variants of a gene, or products of different genes functioning in the same or parallel pathways in an overlapping manner.

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Available from: Jean-Christophe Marine, Sep 29, 2015
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    • "Specific genomic site with high homologous combination frequency and ubiquitous transcriptional activity is also critical for success in gene targeting. The most preferred integration site used for gene targeting is the Rosa26 locus in mouse [18]–[20]. The Rosa26 (Gt(ROSA)26Sor) gene was identified originally as a ubiquitous marker in a retroviral gene-trapping screen in mouse ESCs. "
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    ABSTRACT: Genetically modified pigs have become a popular model system in fundamental research, agricultural and biomedical applications. However, random integration often result in unstable expression of transgene and unpredictable phenotypes. The Rosa26 locus has been widely used to produce genetic modified animals with high and consistent expressing of transgene in mouse, human and rat, as it can be targeted efficiently and is not subject to gene-silencing effects. Recently, the first case of reporter gene targeting pigs in porcine Rosa26 (pRosa26) locus was reported. In the study, full sequence of pRosa26 locus was further characterized, and the pRosa26 promoter (pR26) was cloned and we evidenced that the new porcine endogenous promoter is suitable for driving transgene expression in a high and stable manner by avoiding DNA methylation. Furthermore, elongation factor 1a promoter (EF1a) -driven GFP reporter and Myostatin promoter (MyoP)-driven Follistatin (Fst) were successfully targeted into the pRosa26 locusby traditional homologous recombination (HR) strategy. EF1a showed high activity and hypomethylation at the locus. And, muscle-specific promoter MyoP was activated strictly in muscle of the pRosa26 targeted pigs, indicating Rosa26 locus supports tissue-specific promoter driving transgene expression in its own manner. The study provided further demonstration on biomedical and agricultural applications of porcine Rosa26 promoter and locus.
    PLoS ONE 09/2014; 9(9):e107945. DOI:10.1371/journal.pone.0107945 · 3.23 Impact Factor
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    • "In our studies, cells need to go through multiple experimental manipulations, each of which can diminish such potential. Compared to ES cells derived from inbred strains, F1 hybrid ES cells retain exceptional developmental potential (Eggan et al., 2001), even after multiple rounds of in vitro manipulation such as serial gene targeting (Nyabi et al., 2009), chemical mutagenesis (Chen et al., 2000), and irradiation-induced chromosomal deletion (Chick et al., 2005). As demonstrated here, our hybrid ES cells have gone through targeted manipulations of the Blm gene followed by transposon-mediated gene entrapment and still maintain pluripotency, allowing mouse formation. "
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    ABSTRACT: Longevity is correlated with stress resistance in many animal models. However, previous efforts through the boosting of the antioxidant defense system did not extend life span, suggesting that longevity related stress resistance is mediated by other uncharacterized pathways. We have developed a high-throughput platform for screening and rapid identification of novel genetic mutants in the mouse that are stress resistant. Selection for resistance to stressors occurs in mutagenized mouse embryonic stem (ES) cells, which are carefully treated so as to maintain pluripotency for mouse production. Initial characterization of these mutant ES cells revealed mutations in Pigl, Tiam1, and Rffl, among others. These genes are implicated in glycosylphosphatidylinositol biosynthesis, NADPH oxidase function, and inflammation. These mutants: (1) are resistant to two different oxidative stressors, paraquat and the omission of 2-mercaptoethanol, (2) have reduced levels of endogenous reactive oxygen species (ROS), (3) are capable of generating live mice, and (4) transmit the stress resistance phenotype to the mice. This strategy offers an efficient way to select for new mutants expressing a stress resistance phenotype, to rapidly identify the causative genes, and to develop mice for in vivo studies.
    Frontiers in Genetics 09/2014; 5:310. DOI:10.3389/fgene.2014.00310
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    • "The mouse Rosa26 locus is widely used as a permissive site for targeted placement of transgenes [12], [13], with no detectable effect on animal viability or fertility. ROSA26 homologues have also been identified in rat [14] and human [15]. "
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    ABSTRACT: We are extending the Cre/loxP site-specific recombination system to pigs, focussing on conditional and tissue-specific expression of oncogenic mutations to model human cancers. Identifying the location, pattern and extent of Cre recombination in vivo is an important aspect of this technology. Here we report pigs with a dual fluorochrome cassette under the control of the strong CAG promoter that switches expression after Cre-recombination, from membrane-targeted tandem dimer Tomato to membrane-targeted green fluorescent protein. The reporter cassette was placed at the porcine ROSA26 locus by conventional gene targeting using primary mesenchymal stem cells, and animals generated by nuclear transfer. Gene targeting efficiency was high, and analysis of foetal organs and primary cells indicated that the reporter is highly expressed and functional. Cre reporter pigs will provide a multipurpose indicator of Cre recombinase activity, an important new tool for the rapidly expanding field of porcine genetic modification.
    PLoS ONE 07/2014; 9(7):e102455. DOI:10.1371/journal.pone.0102455 · 3.23 Impact Factor
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