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  • br Results br Discussion TALEs are natural effector proteins

    2018-11-08


    Results
    Discussion TALEs are natural effector proteins secreted by Xanthomonas and Ralstonia bacteria. These proteins regulate gene expression in host plants and thus facilitate bacterial survival and colonization (Moscou and Bogdanove, 2009). TALE contains a DNA-binding domain consisting of 34-amino-acid tandem repeat modules (Moscou and Bogdanove, 2009). Recently, significant efforts have been made toward genetic modification and transcriptional modulation by using TALEs (Cong et al., 2012; Hockemeyer et al., 2011) because designing TALE is much easier than zinc fingers and meganucleases. It was demonstrated previously that the KRAB repressor domain fused with TALE designed specifically to target the promoter of SOX2 is able to inhibit the endogenous expression level of SOX2 (Cong et al., 2012). Loss-of-function studies of miRNAs by the knockout approach are in high demand as these molecules are important gene regulators in multiple biological processes. Conventional knockout strategies are not suitable for deleting miRNAs or miRNA clusters in primary Z-YVAD-FMK manufacturer or cell lines. Here, we knocked out the endogenous miR-302/367 cluster in HFFs by using specific TALENs and a donor vector. Notably, our data showed that almost all the HFFs were homozygous miR-302/367 null mutants after drug selection. We reasoned that the high efficiency of site-specific recombination of the GFP-puromycin expression cassette by the specific TALENs may be due to: i) the use of two, instead of one, pairs of TALENs, and ii) the use of a donor vector with puromycin selection marker. Interestingly, Ansai et al. recently reported that TALENs-induced mutation efficiency is mainly affected by TALENs activity, target sequences, and the amount of TALENs transfected or injected in each cell. They showed that a high concentration of TALENs induced extremely high mutation efficiency (almost 100%), and all the mutants were homozygous (Ansai et al., 2013).
    Experimental Procedures
    Acknowledgments
    Introduction Tauopathies are a group of neurodegenerative disorders characterized by the accumulation and aggregation of the pathological TAU protein in human brains (Hutton, 2000; Lee et al., 2001; Mandelkow and Mandelkow, 2012; Vossel and Miller, 2008). TAU, encoded by the MAPT gene on chromosome 17, is a microtubule binding protein that is highly expressed in neurons and predominantly located in neuronal axons (Goedert et al., 1989; Kosik et al., 1989; Morris et al., 2011; Neve et al., 1986). Hyperphosphorylation and aggregation of TAU cause neurofibrillary pathologies, including tangles and neuropil threads that are often seen in brains of patients with Alzheimer’s disease (AD) (Hutton, 2000; Kosik et al., 1986; Lee et al., 2001; Mandelkow and Mandelkow, 2012; Vossel and Miller, 2008). Tauopathies are also typical of other neurodegenerative disorders, including frontotemporal dementia (FTD) and progressive supranuclear palsy (PSP) (Hutton, 2000; Lee et al., 2001; Mandelkow and Mandelkow, 2012; Vossel and Miller, 2008). Most FTD and PSP cases are sporadic, but a minority are familial (Weder et al., 2007). FTD and PSP syndromes can be caused by mutations in the MAPT gene that result in abnormal TAU phosphorylation and aggregation, leading to neurodegeneration. Transgenic murine models of tauopathies have revealed fundamental insights into the disease (Lee et al., 2001; Mandelkow and Mandelkow, 2012), but their value as predictive preclinical models is unknown. In fact, many candidate drugs successful in rodent models of neurodegenerative diseases have failed in humans (Ashe and Zahs, 2010; Huang and Mucke, 2012). Thus, new humanized disease models, such as mutation- and patient-specific induced pluripotent stem cells (iPSCs), are urgently needed for further development of therapeutic strategies for tauopathies. Human iPSCs are a highly promising approach for investigating cellular properties of traditionally challenging neurodegenerative disorders (HD iPSC Consortium, 2012; Israel et al., 2012; Kondo et al., 2013; Park et al., 2008; Yamanaka, 2009). Because postmortem tissue represents late-stage disease, modeling tauopathy onset and progression are problematic with autopsy samples alone. iPSC-derived neurons grown in culture allow for the detection of specific molecular and temporal signatures from mutation-carrying patients, thereby improving our understanding of the pathogenesis of tauopathies.