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    • Targeting experiments with a second eGFP reporter cell line

    2018-11-08

    Targeting experiments with a second eGFP reporter cell line (hCBiPS2eGFPC16) showed similar results. The observed 3-fold lower eGFP gene targeting efficiency for hCBiPS2eGFPC16 as compared with hCBiPS2eGFPC7 cells most likely represents clone-specific variations with respect to survival rates after single-cell dissociation, transfection efficiency, and general growth characteristics under feeder-free monolayer culture conditions. Correct targeting in eGFPnegRedStarpos cells was confirmed through PCR and Southern blot analysis in 39 out of 41 isolated clones derived from two targeting experiments. In the two remaining clones, accurate targeting occurred with respect to the 5′ junction, but the 3′ junction was not correctly formed. As observed for the established eGFP reporter clone, RedStar expression of different targeted clones was almost uniform and persisted to a large extent during differentiation. Subsequently, we aimed to evaluate gene targeting for another genomic locus. Through a highly efficient transfection of AAVS1-specific ZFN and TALEN plasmids, we obtained targeting efficiencies that were similar, or in the case of TALENs even higher, than the 0.8% reported by Lombardo et al. (2011) for eGFP integration in the same locus using integrase-deficient lentiviral ZFN vectors. In the case of plasmid-donor-based AAVS1 targeting, transgenic clones were identified based on the emergence of eGFP or RedStar expression. Remarkably, only four nontargeted clones among a total of 120 isolated eGFPpos or RedStarpos clones were detected by PCR. This high proportion of ∼97% correctly targeted clones, together with the fact that on average only 40% of correctly targeted clones contained additional random integrations, underlines the efficiency of ZFN-based gene targeting in our protocol. Notably, the frequency of additional (nontargeted) integration was in a range similar to the 24%–38% reported in previous studies that applies transgene-based antibiotic selection after ZFN- or TALEN-mediated integration at AAVS1 in hiPSCs (Hockemeyer et al., 2009, 2011; Zou et al., 2009). Interestingly, compared with these studies, which reported 6%–32% biallelic targeting (Hockemeyer et al., 2009; Zou et al., 2011), we observed an even higher proportion (13%–75%) of ZFN-targeted clones in which both ibuprofen msds had been edited correctly. Given that correct targeting occurs only in up to 0.8% of transfected cells, one would actually expect a much lower frequency of biallelic targeting. The unexpectedly high proportion of biallelic targeting events observed suggests that in the majority of cells, the intracellular conditions do not allow for efficient targeting, whereas the generation of DSBs and subsequent repair through HR is highly efficient in only a relatively small proportion of cells, resulting in a considerable number of biallelic targeting events. These conditions may include efficient transfection of the respective cell, the appropriate timing of the peak of ZFN expression and adequate cell-cycle phases (S or G2 phase for HR), and the simultaneous expression of essential HR cofactors. Finally, it is worth mentioning that our targeting protocol did not affect the pluripotent state of the resulting clones, as indicated by the persistent expression of typical pluripotency markers and efficient embryoid body (EB)-based differentiation into derivatives of all three germ layers. Moreover, we were able to exclude any karyotypic abnormalities in our analyzed cell clones and also obtained robust transgene expression from the AAVS1 locus upon differentiation, indicating that ZFN/TALEN treatment and application of a ROCK inhibitor (despite its potential side effects; Chapman et al., 2010) did not affect or impair the PSC properties. Interestingly, we obtained similar targeting efficiencies using TALENs in combination with short ssODNs with 2 × 43 bp of homology to the AAVS1 locus instead of plasmid donors with 2 × 700 bp of homology. Here, we assume that the lower pairing probability of a relatively short homologous oligonucleotide sequence is compensated for by the much higher number of molecules that enter the transfected cells in comparison with a 5.9 kb plasmid donor. The calculated high targeting frequency of ∼1.6% enabled us to identify correctly targeted single-cell clones after transfection and limiting dilution simply by PCR. To date, the insertion of ssODNs on a clonal level has only been reported by Soldner et al. (2011) and Ding et al. (2013), who introduced point mutations in hESCs. However, in contrast to our study, both of these groups preselected positive transfected cells and subsequently established clones with efficiencies of 0.4%–0.8% (Soldner et al., 2011) and 1.5% (Ding et al., 2013) from the preselected cell population.