When reverse genetics meets physiology: The use of site-specific recombinases in mice

Columbia University, New York, New York, United States
FEBS Letters (Impact Factor: 3.17). 11/2002; 529(1):116-21. DOI: 10.1016/S0014-5793(02)03266-0
Source: PubMed


The use of site-specific recombinases enables the precise introduction of defined genetic mutations into the mouse genome. In theory, any deletion, point mutation, inversion or translocation can be modeled in mice. Because gene targeting is controlled both spatially and temporally, the function of a given gene can be studied in the desired cell types and at a specific time point. This 'genetic dissection' allows to define gene function in development, physiology or behavior. In this review, we focus on the technical possibilities of Cre and other site-specific recombinases but also discuss their limitations.

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Available from: Emilio Casanova, Dec 16, 2013
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    • "AAV integration into somatic genomes is well documented (Smith, 2008) and Cre-lox can mediate integration reversibly with excision (Tronche et al., 2002). The robust drop in reporter fluorescence suggests a highly efficient and irreversible process, but how DO-GFP integration could become permanent is unclear. "
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    ABSTRACT: Understanding the organization of the nervous system requires methods for dissecting the contributions of each component cell type to circuit function. One widely used approach combines genetic targeting of Cre recombinase to specific cell populations with infection of recombinant adeno-associated viruses (rAAVs) whose transgene expression is activated by Cre ("Cre-On"). Distinguishing how the Cre-expressing neurons differ functionally from neighboring Cre-negative neurons requires rAAVs that are inactivated by Cre ("Cre-Off") and can be used in tandem with Cre-On viruses. Here we introduce two rAAV vectors that are inactivated by Cre and carry different fluorophore and optogenetic constructs. We demonstrate single and dual rAAV systems to achieve Cre-On and Cre-Off expression in spatially-intermingled cell populations of the striatum. Using these systems, we uncovered cryptic genomic interactions that occur between multiple Cre-sensitive rAAVs or between Cre-sensitive rAAVs and somatic Cre-conditional alleles and devised methods to avoid these interactions. Our data highlight both important experimental caveats associated with Cre-dependent rAAV use as well as opportunities for the development of improved rAAVs for gene delivery.
    Frontiers in Neural Circuits 07/2012; 6:47. DOI:10.3389/fncir.2012.00047 · 3.60 Impact Factor
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    • "The Cre recombinase is an enzyme derived from the bacteriophage P1 that catalyzes the recombination between two 34 bp sequences named loxP (Abremski et al., 1983). The Cre/loxP system can be used to inactivate or activate a gene in a cell type in a temporally-controlled manner (Branda and Dymecki, 2004; Kwan, 2002; Tronche et al., 2002). The Cre/loxP system is most commonly applied by combination of two elements: a transgenic line expressing the Cre recombinase under a tissue specific promoter and a second transgenic line harboring a gene of interest which is flanked by two loxP sites. "
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    ABSTRACT: Here we describe the generation of an inducible Cre transgenic line allowing conditional mutagenesis in ovarian granulosa cells. We have expressed the tamoxifen inducible CreER(T)² fusion protein from a Bacterial Artificial Chromosome (BAC) containing the regulatory elements of the hydroxysteroid (17-beta) dehydrogenase 1 (Hsd17b1) gene. Hsd17b1-iCreER(T)² transgenic mice express the iCreER(T)² fusion protein exclusively in ovarian granulosa cells. Recombination analysis at the genomic DNA level using mice with "floxed" Stat3 alleles showed no Cre activity in absence of tamoxifen whereas tamoxifen treatment induced Cre activity solely in the ovaries. Further characterization of Hsd17b1-iCreER(T)² mice using a Cre reporter line demonstrated that Cre-mediated recombination was restricted to ovarian granulosa cells. Therefore, Hsd17b1-iCreER(T)² mice should be a useful tool to analyze the gene functions in ovarian granulosa cells.
    genesis 10/2010; 48(10):612-7. DOI:10.1002/dvg.20664 · 2.02 Impact Factor
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    • "Just 67 bp of cis-acting sequences (RSS12 + RSS23) need to be provided to ensure correct expression of the exogenous gene (unpublished results). The ability to switch gene expression from one gene to another by harnessing endogenous recombinase activity may have practical applications in the B and T cell lineage analogous to the use of the Cre/Lox system or other exogenous recombinases [19]. "
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    ABSTRACT: Diversity of immunoglobulins and the T cell antigen receptors is achieved via the recombination activating gene (RAG)-mediated rearrangement of variable (V), diversity (D) and joining (J) gene segments, and this underpins the efficient recognition of a seemingly limitless array of antigens. Analysis of V(D)J recombination activity is typically performed using extrachromosomal recombination substrates that are recovered from transfected cells and selected using bacterial transformation. We have developed a two-colour fluorescence-based system that simplifies detection of both deletion and inversion joining events mediated by RAG proteins. This system employs two fluorescent reporter genes that differentially mark unrearranged substrates and those that have undergone RAG-mediated deletion or inversion events. The recombination products bear the hallmarks of true V(D)J recombination and activity can be detected using fluorescence microscopy or flow cytometry. Recombination events can be detected without the need for cytotoxic selection of recombination products and the system allows analysis of recombination activity using substrates integrated into the genome. This system will be useful in the analysis and exploitation of the V(D)J recombination machinery and suggests that similar approaches could be used to replace expression of one gene with another during lymphocyte development.
    Mobile DNA 03/2010; 1(1):9. DOI:10.1186/1759-8753-1-9 · 2.11 Impact Factor
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