Our findings indicate that rat

Our findings indicate that rat ESCs are more sensitive to GSK inhibition primarily because of the relative abundance of LEF1, which favors the activation of differentiation genes. Their collective expression may challenge and destabilize the self-renewal circuitry. The TOPFlash assays also suggest that there may be higher basal levels of intracellular β-catenin in rat ESCs. This may contribute to the reduced threshold of GSK3 inhibition for full derepression of Esrrb. Rat ESCs cultured in T2iL retain competence to form chimeras and give germline transmission, even after clonal expansion, with at least comparable efficiency to previous reports from our group and others using 2iL (Blair et al., 2012; Tong et al., 2010). By reducing differentiation, culture in T2iL may offer a more robust platform for expansion and genetic manipulation. However, T2iL does not improve karyotype stability, implying that there is additional selection pressure in the present culture milieu.
Experimental Procedures
Acknowledgments
Introduction MicroRNAs (miRNAs) are small 18–24 nt single-stranded noncoding RNA molecules that regulate gene expression at the posttranscriptional level. These small RNAs bind to partially complementary target sites in messenger RNAs (mRNAs), leading to degradation of target mRNAs or translational repression of encoded proteins (Bartel, 2004). To date, there are 1281 and 2042 mature miRNAs in mouse and human genomes, respectively. These miRNAs are implicated in many biological processes, diseases, development, and cellular reprogramming (Bartel, 2004; Johnston and Hobert, 2003). Several approaches for knockdown of miRNAs have been extensively used, such as locked nucleic Z-YVAD-FMK (LNA) oligonucleotides, antagomirs, and miRZip inhibitors (Liao et al., 2011; Robertson et al., 2010; Xia et al., 2012). Unfortunately, LNA and antagomirs can only transiently inhibit miRNA function. In contrast, miRZips are stably expressed RNAi hairpins and can permanently inhibit miRNA function; however, their inhibitory efficiency, similar to that of LNA and antagomir, is dependent on their dosage in each cell. In addition, the specificity of these three miRNA inhibitors is inversely proportional to their dosage delivered in cells. Therefore, there are significant concerns regarding the specificity and potency of these miRNA inhibitors (Robertson et al., 2010). To overcome these obstacles, we describe two approaches for studying miRNA functions: (1) TALE-based transcriptional repressor for knockdown of miRNA clusters, and (2) TALENs for knockout of miRNA clusters. Originally found to be secreted by Xanthomonas and Ralstonia bacteria, TALE is protein consisting of multiple repeated, highly conserved 33–34 amino acid sequences (Moscou and Bogdanove, 2009), which can be quickly engineered to bind virtually any desired DNA sequence. Thus, TALE can regulate expression of endogenous genes when tethered with transcription activator or repressor domains and edit the genome when fused with the FokI cleavage domain. TALEN is an emerging technology for genome editing (Hockemeyer et al., 2011). In the current study, we applied these two approaches to study the roles of the endogenous miR-302/367 cluster in cellular reprogramming. The miR-302/367 cluster is a polycistronic miRNA consisting of five mature miRNAs (miR-302b/c/a/d and miR-367). It has been well documented that overexpression of this cluster promotes cellular reprogramming and maintains the stemness of human embryonic stem cells (hESCs) (Lin et al., 2011; Miyoshi et al., 2011; Rosa et al., 2009). However, it remains unknown whether the endogenous miR-302/367 cluster is required for the generation of induced pluripotent stem cells (iPSCs). In this study, we efficiently knocked down the expression of mature miR-302/367 miRNAs using the TALE-based transcriptional repressor and deleted the entire miRNA cluster by TALENs to investigate the roles of this cluster in cellular reprogramming.