In addition to configurations for constitutive gene expressi

In addition to configurations for constitutive gene expression, we also tested the behavior of the Tet-on system (http://www.clontech.com) in which sequences encoding either GFP or mCherry expression were placed under the control of a doxycycline-responsive promoter. Although robust doxycycline-inducible expression was observed in early passage lines, the vectors eventually underwent silencing. It is not clear whether this silencing was an inherent property of the genomic position of the transgene or a function of the vector configuration. Whatever the underlying mechanism, silencing or variegated expression of transgenes that contain either inducible or tissue-specific promoters seems to be an issue affecting other “safe harbor” loci (Ordovas et al., 2015). Taking into account the 3D structure of GAPDH, we hypothesized that the presence of 2A peptide at the C terminus would not interfere with GAPDH function. However, western blot analysis indicated a clear reduction in the level of GAPDH-T2A protein relative to GAPDH protein translated from the unmodified GAPDH allele. Analysis of different vector configurations suggested that this lower level of expression was in part due to sequences downstream of the reporter gene. We observed that expression of GFP from different variants of the GT vectors differed substantially, depending on the sequence of the selectable marker 3′ of the IRES. Indeed, in vectors that lacked the IRES-selectable marker cassette altogether, the intensity of GFP was almost ten times brighter than in glucocorticoid receptor antagonist containing a GT vector that included the IRES-Mygro cassette (Figure S1E). However, all PSCs containing the GT vector efficiently differentiated into a variety of lineages, suggesting that the presence of the genetic modification did not impinge on differentiation potential.
Experimental Procedures
Author Contributions
Acknowledgments The authors thank Matt Burton and Paul Lau for help with flow cytometry and Katerina Vlahos for assistance with teratoma experiments. This work was supported by the Victorian Government\'s Operational Infrastructure Support Program and Australian Government National Health and Medical Research Council Independent Research Institute Infrastructure Support Scheme (NHMRC IRIISS), Stem Cells Australia, the NHMRC, and the Victoria-California Stem Cell Alliance. We are also grateful for the generous support of the Stafford Fox Foundation. D.J.A. was supported by the European Union\'s Seventh Framework Program (FP7/2007-2013) under grant agreement PIOF-GA-2010-276186.). C.L.P. was supported by a Viertel Senior Medical Research Fellowship, Australia. A.G.E. and E.G.S. are Senior Research Fellows of the NHMRC. S.E.H. is supported by an NHMRC Overseas Biomedical Fellowship.
Introduction Human pluripotent stem cells (hPSCs) enable modeling aspects of development and disease, and hold great promise for regenerative medicine and drug discovery (van Hoof et al., 2012; Young, 2011). Previous large-scale analyses of hPSCs shed light on pluripotency, differentiation, and de-differentiation by focusing on transcriptional regulation, epigenetic changes, and non-coding RNAs (Boyer et al., 2005; Brandenberger et al., 2004; Elkabetz et al., 2008; Martinez and Gregory, 2010). However, proteomes contain vast amounts of biological information unobtainable via genomics, transcriptomics, or similar analyses (Wilhelm et al., 2014). Thus, a detailed characterization of pluripotency, lineage specification, and reprogramming by protein profiling is important for complementing other analytical methods and should help to elucidate novel mechanisms. Regulation of proteins includes quantitative changes and post-translational modifications (PTMs) (Huttlin et al., 2010). A key PTM is reversible phosphorylation of serine (pS), threonine (pT), and tyrosine (pY), which modulates enzyme activities, protein-protein interactions, conformational changes, protein half-life, and signal transduction, among others (Choudhary and Mann, 2010). Multidimensional liquid chromatography (MDLC) coupled with tandem mass spectrometry (MS/MS) enables large-scale analysis of proteomes and phosphoproteomes (Huttlin et al., 2010; Sharma et al., 2014). Although previous reports have provided important insights into the proteomes of hPSCs (Brill et al., 2009; Munoz et al., 2011; Phanstiel et al., 2011; Rigbolt et al., 2011; Swaney et al., 2009; Van Hoof et al., 2009; Van Hoof et al., 2006), none of these studies have applied robustly controlled differentiation strategies in feeder-free monolayer cultures. Hence, proteomic analysis of pluripotent cells compared with their lineage-specific multipotent derivatives has not been reported. Moreover, previous datasets did not reach the depth enabled by recent technical advances (Huttlin et al., 2010; Sharma et al., 2014). Notably, label-free quantification (LFQ) can yield deeper proteome coverage than stable-isotope labeling by amino acids in cell culture while maintaining quantitative accuracy (Collier et al., 2010; Gokce et al., 2011; Sharma et al., 2014).