Schnutgen, F. et al. Genomewide production of multipurpose alleles for the functional analysis of the mouse genome. Proc. Natl. Acad. Sci. USA 102, 7221-7226

Department of Molecular Hematology, University of Frankfurt Medical School, 60590 Frankfurt am Main, Germany.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 06/2005; 102(20):7221-6. DOI: 10.1073/pnas.0502273102
Source: PubMed


A type of retroviral gene trap vectors has been developed that can induce conditional mutations in most genes expressed in mouse embryonic stem (ES) cells. The vectors rely on directional site-specific recombination systems that can repair and re-induce gene trap mutations when activated in succession. After the gene traps are inserted into the mouse genome, genetic mutations can be produced at a particular time and place in somatic cells. In addition to their conditional features, the vectors create multipurpose alleles amenable to a wide range of post-insertional modifications. Here we have used these directional recombination vectors to assemble the largest library of ES cell lines with conditional mutations in single genes yet assembled, presently totaling 1,000 unique genes. The trapped ES cell lines, which can be ordered from the German Gene Trap Consortium, are freely available to the scientific community.

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    • "Regulated expression of small hairpin RNA has also been used for reversible gene repression, but the repression is usually incomplete [2]. Finally, transcription stop sequences or gene-trap cassettes, which are removable/inactivable, can be inserted into target genes, leading to constitutive KO that can be conditionally rescued, but this strategy is not suitable for conditional induction of gene KO [1,3,10,11]. "
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    ABSTRACT: Background Conditional gene knockout (cKO) mediated by the Cre/LoxP system is indispensable for exploring gene functions in mice. However, a major limitation of this method is that gene KO is not reversible. A number of methods have been developed to overcome this, but each method has its own limitations. Results We describe a simple method we have named LOFT [LoxP-flippase (FLP) recognition target (FRT) Trap], which is capable of reversible cKO and free of the limitations associated with existing techniques. This method involves two alleles of a target gene: a standard floxed allele, and a multi-functional allele bearing an FRT-flanked gene-trap cassette, which inactivates the target gene while reporting its expression with green fluorescent protein (GFP); the trapped allele is thus a null and GFP reporter by default, but is convertible into a wild-type allele. The floxed and trapped alleles can typically be generated using a single construct bearing a gene-trap cassette doubly flanked by LoxP and FRT sites, and can be used independently to achieve conditional and constitutive gene KO, respectively. More importantly, in mice bearing both alleles and also expressing the Cre and FLP recombinases, sequential function of the two enzymes should lead to deletion of the target gene, followed by restoration of its expression, thus achieving reversible cKO. LOFT should be generally applicable to mouse genes, including the growing numbers of genes already floxed; in the latter case, only the trapped alleles need to be generated to confer reversibility to the pre-existing cKO models. LOFT has other applications, including the creation and reversal of hypomorphic mutations. In this study we proved the principle of LOFT in the context of T-cell development, at a hypomorphic allele of Baf57/Smarce1 encoding a subunit of the chromatin-remodeling Brg/Brahma-associated factor (BAF) complex. Interestingly, the FLP used in the current work caused efficient reversal in peripheral T cells but not thymocytes, which is advantageous for studying developmental epigenetic programming of T-cell functions, a fundamental issue in immunology. Conclusions LOFT combines well-established basic genetic methods into a simple and reliable method for reversible gene targeting, with the flexibility of achieving traditional constitutive and conditional KO.
    BMC Biology 11/2012; 10(1):96. DOI:10.1186/1741-7007-10-96 · 7.98 Impact Factor
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    • "Site specific recombinases (SSRs) are available for efficient targeted transgene insertion into the genome. Especially, the use of the Cre and Flp SSRs in recombinase-mediated cassette exchange (RMCE) has proven efficient for transgene targeting in the mouse genome (Osterwalder et al. 2010; Schnutgen et al. 2005; Cobellis et al. 2005; Schebelle et al. 2010). The Sleeping Beauty (SB) DNA transposon system is well established in the mouse (Dupuy et al. 2001, 2005; Carlson et al. 2003, 2005, 2011b; Mates et al. 2009; Kitada et al. 2007; Geurts et al. 2006) and has been used to transfer transgenes into the genome of porcine cells (Clark et al. 2007; Jakobsen et al. 2011a, b; Carlson et al. 2011a). "
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    ABSTRACT: Targeted transgenesis using site-specific recombinases is an attractive method to create genetically modified animals as it allows for integration of the transgene in a pre-selected transcriptionally active genomic site. Here we describe the application of recombinase-mediated cassette exchange (RMCE) in cells from a Göttingen minipig with four RMCE acceptor loci, each containing a green fluorescence protein (GFP) marker gene driven by a human UbiC promoter. The four RMCE acceptor loci segregated independent of each other, and expression profiles could be determined in various tissues. Using minicircles in RMCE in fibroblasts with all four acceptor loci and followed by SCNT, we produced piglets with a single copy of a transgene incorporated into one of the transcriptionally active acceptor loci. The transgene, consisting of a cDNA of the Alzheimer’s disease-causing gene PSEN1M146I driven by an enhanced human UbiC promoter, had an expression profile in various tissues similar to that of the GFP marker gene. The results show that RMCE can be done in a pre-selected transcriptionally active acceptor locus for targeted transgenesis in pigs. Electronic supplementary material The online version of this article (doi:10.1007/s11248-012-9671-6) contains supplementary material, which is available to authorized users.
    Transgenic Research 10/2012; 22(4). DOI:10.1007/s11248-012-9671-6 · 2.32 Impact Factor
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    • "Beside the disruption of transcriptionally silent genes in the target cells, another challenge for random gene trapping has been the conditional inactivation of identified genes (16,19–21). To achieve this in poly(A) trapping, we assembled critical components of a gene-trap vector, as indicated in Figure 2. The first half represents a gene-terminator cassette containing a promoterless enhanced green fluorescent protein (EGFP) cDNA for monitoring the expression of trapped genes in living cells (7–9) and two or four copies of poly(A)-addition signals for the complete transcriptional termination. "
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    ABSTRACT: Among the insertional mutagenesis techniques used in the current international knockout mouse project (KOMP) on the inactivation of all mouse genes in embryonic stem (ES) cells, random gene trapping has been playing a major role. Gene-targeting experiments have also been performed to individually and conditionally knockout the remaining 'difficult-to-trap' genes. Here, we show that transcriptionally silent genes in ES cells are severely underrepresented among the randomly trapped genes in KOMP. Our conditional poly(A)-trapping vector with a common retroviral backbone also has a strong bias to be integrated into constitutively transcribed genome loci. Most importantly, conditional gene disruption could not be successfully accomplished by using the retrovirus vector because of the frequent development of intra-vector deletions/rearrangements. We found that one of the cut and paste-type DNA transposons, Tol2, can serve as an ideal platform for gene-trap vectors that ensures identification and conditional disruption of a broad spectrum of genes in ES cells. We also solved a long-standing problem associated with multiple vector integration into the genome of a single cell by incorporating a mixture of differentially tagged Tol2 transposons. We believe our strategy indicates a straightforward approach to mass-production of conditionally disrupted alleles for genes in the target cells.
    Nucleic Acids Research 03/2012; 40(13):e97. DOI:10.1093/nar/gks262 · 9.11 Impact Factor
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