br Design of donor plasmids If no

Design of donor plasmids If no footprintless gene targeting is required or it is intended to introduce larger transgenes, donor plasmids have to be constructed that contain the homology arms flanking the transgenes or selection cassettes coupled to promoters of choice. Similarly, donor plasmids can be constructed to enable expression of a functional protein under control of the natural endogenous locus. Clearly, the design of the donor plasmid is of utmost importance for the success of the targeting as well as the proper expression of the inserted genes. If possible, the homology arms of the targeting vector should exactly match the individual targeting sequence and should not be more than 40bp away from the double strand break site (Stark et al., 2004). Longer distances can also work but will result in lower efficiency of HR, which can be compensated for by the application of antibiotic selection cassettes. We typically use homology arms as close as possible to the break site and homology arms of about 500–800bp on each site to insert up to 9kb into the genomic target site. In general, longer homology arms may increase the chance for pairing with corresponding genomic sequences but on the other hand lead to an increase in donor plasmid size and lower transfection rates (see ‘Techniques to achieve appropriate expression of designer nucleases in pluripotent stem cells’). For the application of constitutive promoters in undifferentiated human PSCs as well as in differentiated derivatives, we recommend the CAG or PGK promoter, keeping in mind that the decision for an appropriate poly A signal is also of utmost importance for reliable gene expression. In our experience the RBG, BGH or HSV-TK poly A sites are working properly in hPSCs and also in their differentiated counterparts. In general, viral-derived elements like the CMV promoter or SV40 poly A sites work less well in hPSCs and should be avoided if proper expression in undifferentiated plk1 inhibitor is intended. In order to minimize the plasmid size, in case of simultaneous expression of more than one gene, we suggest expression under control of a joint promoter through a short self-cleaving 2A peptide instead of using an internal ribosomal entry site (IRES), which is usually longer than 500bp. In addition, the 2A peptide enables stoichiometric expression of multiple proteins, whereas the translation efficiency of a gene behind an IRES is much lower compared to the gene in front (Ibrahimi et al., 2009; Kim et al., 2011).
Pluripotent stem cell culture conditions and single cell cloning
Conclusions
Acknowledgments This work was funded by the German Federal Ministry of Education and Research (CARPuD, 01GM111A), the German Center for Lung Research (DZL, 82DZL00201), the German Research Foundation (Cluster of Excellence REBIRTH, EXC 62/3) and the Mukoviszidose Institut GmbH (S03/11). We thank R. Diestel and M. Coffee for their critical readings of the manuscript.
Introduction Radiation therapy is critical in the treatment of brain tumors such as glioblastoma multiforme (Stupp et al., 2009; Stupp et al., 2005; Kumabe et al., 2013; DeAngelis, 2005; Rusthoven et al., 2014; Chen et al., 2013). Modern techniques such as intensity-modulated radiation therapy allow focused delivery of radiation dose to the tumor while minimizing radiation dose to the adjacent critical structures. Nonetheless, adjacent healthy brain tissue also receives some radiation dose during treatment depending on the tumor location and geometry. Cellular and functional effects have been associated with radiation to the neurogenic niches (Achanta et al., 2009; Tada et al., 2000; Crossen et al., 1994; Monje et al., 2002; Capilla-Gonzalez et al., 2014; Padovani et al., 2012; Armstrong et al., 2013). The subventricular zone (SVZ) of the lateral ventricles and the dentate gyrus (DG) of the hippocampus constitute the main neurogenic niches of the adult mammalian brain (Gates et al., 1995; Alvarez-Buylla et al., 2002; Doetsch et al., 1997; Seri et al., 2001; Eriksson et al., 1998; Quinones-Hinojosa et al., 2006). Cranial radiation is known to inhibit proliferation and neurogenesis in the hippocampus, which has been related to learning and memory deficits in rodents and humans (Achanta et al., 2009; Tada et al., 2000; Monje et al., 2002; Padovani et al., 2012; Armstrong et al., 2013; Redmond et al., 2013; Monje, 2008; Sato et al., 2013; Calabrese et al., 2009; Marazziti et al., 2012; Raber et al., 2004). Similarly, radiation of the rodent SVZ depletes precursor cells and decreases the production of new cells, affecting the consolidation and restitution of olfactory traces in the olfactory bulb (OB) (Balentova et al., 2013; Lazarini et al., 2009; Achanta et al., 2012), as well as the ability of the SVZ to respond to brain damage (Capilla-Gonzalez et al., 2014). Despite these negative effects, retrospective data suggest a potentially prolonged overall survival in patients with glioblastoma that received high dose of ipsilateral SVZ radiation(Chen et al., 2013; Gupta et al., 2012; Kast et al., 2013; Evers et al., 2010; Lee et al., 2013a; Lee et al., 2013b; Chen et al., 2015). In line with these reports, a prospective study of hypofractionated radiation therapy found improved survival in long term survivors with necrosis in the SVZ (Iuchi et al., 2014). In this review, we highlight the current knowledge regarding the cellular and functional effects of SVZ radiation, focusing in its implication on brain tumor therapy.