The Bag KO mESC line

The Bag1KO-mESC line maintains a classical dome-shaped ES cell morphology and expressed high levels of ES-associated protein such as, alkaline phosphatase (Fig. 1D). Immunofluorescent staining for the three key pluripotent stem cell markers Oct4, Sox2 and Nanog revealed that the Bag1KO-mESC line was positive for these all three stemness markers (Fig. 1E). Karyotype analysis also confirmed that the CRISPR/Cas9 gene editing process did not introduce any chromosomal abnormality in the Bag1KO-mESC line (Fig. 1F). Furthermore, the Bag1KO-mESC line still maintained the potential to differentiate into all three germ layers as determined by their ability to form teratoma in vivo and histological examinations (Fig. 1G). In summary, our Bag1KO-mESC line has been determined to be pluripotent and possess a normal karyotype. This mutant cell line would be a good model for investigating the biological function of Bag1 during development.
Materials and methods
Conflict of interest
Acknowledgements This work was supported by Research Grant Council CUHK14100315.
Resource table Resource details Blastocysts (E4.5) from the crossing of heterozygous Sprague-Dawley (SD) GFP rats (SD-Tg(GFP)1BalRrrc) and from wild type Wistar-Kyoto (WK) rats were used to isolate embryonic stem (ES) pgi2 in N2B27 2i media. In this report we present the detailed characterization of one cell line from each rat strain: ATCe-rSD78 from SD strain and ATCe-rWK1 from WK strain. SD-Tg(GFP)1BalRrrc rats express the GFP under the control of the human ubiquitin promoter. Nevertheless, we observed that isolated ES cells did not show homogeneous GFP fluorescence signal and the penetrance of the GFP was variable in the different cell lines, therefore we selected the GFP-negative cell line ATCe-rSD78 and both ATCe-rSD78 and ATCe-rWK1 were transduced with pWPXL lentiviral construct driving eGFP expression under the control of the Ef1α promoter. Cells were transduced at early passages, FACS sorted, expanded and characterized. Both cell lines were splitted 1:3–1:4 every 3–5days and displayed a typical small, round shape and tightly packed colony morphology (Fig. 1A). Cells were routinely tested for mycoplasma contamination and microsatellite analysis confirmed the different origin of the two cell lines (Supplementary Fig. 1). Both cell lines show normal karyotype in more than 80% of the cells, which is in accordance with previous publications related to rat ES cells isolation (Li et al. 2008), (Fig. 1B and Table 1). The expression of several pluripotency-associated markers such as OCT3/4, NANOG, SOX2, KLF4, ESRRB1, SSEA-1 was confirmed by qRT-PCR (Fig. 1C) and/or cell staining (Fig. 1D). SSEA-4, a primate (primed) pluripotent stem cells marker was not expressed, as expected. Additionally, cells possess alkaline phosphatase (AP) activity (Fig. 1E). To assay differentiation potential we performed the in vivo teratoma-formation assay in Rag2−/− ƔC−/− immunodeficient mice. After 4weeks, formed teratomas were excised and analyzed histologically, revealing presence of differentiated tissues derived from the three germ layers (Fig. 1F). By immunofluorescence staining we confirmed the expression of GFP reporter in differentiated cells from the three germ layers within the teratoma (Fig. 1G).
Materials and methods
Author disclosure statement
Resource table Resource details Urinary cells of a healthy male aged 38years were obtained and induced to hiPSCs using Sendai virus based non-integration vectors. Morphologies of donor cells and generated hiPSCs, KSCBi001-A are shown in Fig. 1A. Pluripotency of generated hiPSCs, KSCBi001-A was characterized by immunofluorescence staining markers for Oct4, SSEA4, Tra-1-60, and Tra-1-81 (Fig. 1B). KSCBi001-A has a normal karyotype (46,XY) analyzed by GTG-banding with resolution of 550 bands (Fig. 1C). All Sendai viral transgenes were silenced in KSCBi001-A as analyzed by RT-PCR using Sendai viral specification markers (Fig. 1D). Differentiation capacity of KSCBi001-A into all three germ layers was confirmed by real-time PCR using germ layer specification markers for Pax6 (ectoderm), AFP (endoderm), and T (mesoderm) in KSCBi001-A-derived embryoid bodies (Fig. 1E–F). All 16 allele loci of KSCBi001-A are consistent with donor cells, as confirmed by STR analysis (Fig. 1G).