We used two mouse embryonic fibroblast (MEF) fractions and two tail-tip fibroblast (TTF) fractions as the parent somatic cells, and each was prepared from an independent single embryo or adult. The cell lines used in this study were generated from the C57BL/6J mouse strain (Japan SLC, Hamamatsu, ) except for a set of three ntES sister lines that were generated from a C57BL/6N mouse (Japan SLC). Materials and Methods Mouse Strains and Parent Somatic Cells Furthermore, we explored whether it would be possible to reduce the point mutation frequency during genome reprogramming. We have here studied another type of genome reprogrammed pluripotent stem cells, nuclear transfer embryonic stem cells (ntESCs), to address whether point mutations generally arise during genome reprogramming in these cells. Therefore, in addition to the study of the molecular mechanisms underlying these genetic aberrations, the issue whether the point mutation frequency in iPSCs can be reduced has become a focus of attention. Whereas induced pluripotent stem cells (iPSCs) hold great promise for use in regenerative medicine 1- 8, the identification of a substantial number of point mutations in their genomes 9- 15 has aroused concerns that they may not be safe to use, since such mutations could have tumorigenic or immunogenic effects 16- 19. Importantly, our results suggest that a greatly reduced point mutation frequency in induced pluripotent stem cells is feasible by optimizing various genome reprogramming conditions. Our present results thus show that point mutations in iPS cells are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. We thereby succeeded in establishing ntES cell lines with far fewer point mutations. We conducted whole genome sequencing of nuclear transfer ES cells derived from MEFs or tail-tip fibroblasts (TTFs). IPS cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Base substitution profile analysis confirmed this greatly reduced number of point mutations. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Error probabilities.Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. Įwing B, Green P (1998) Base-calling of automated sequencer traces using phred. Koch CM, Chiu SF, Akbarpour M, Bharat A, Ridge KM, Bartom ET, Winter DR (2018) A Beginner’s guide to analysis of RNA sequencing data. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-seq. īyron SA, Van Keuren-Jensen KR, Engelthaler DM, Carpten JD, Craig DW (2016) Translating RNA sequencing into clinical diagnostics: opportunities and challenges. Ozsolak F, Milos PM (2011) RNA sequencing: advances, challenges and opportunities. Royce TE, Rozowsky JS, Gerstein MB (2007) Toward a universal microarray: prediction of gene expression through nearest-neighbor probe sequence identification. Okoniewski MJ, Miller CJ (2006) Hybridization interactions between probesets in short oligo microarrays lead to spurious correlations. Van Hal NL, Vorst O, van Houwelingen AM, Kok EJ, Peijnenburg A, Aharoni A, van Tunen AJ, Keijer J (2000) The application of DNA microarrays in gene expression analysis. Wang Z, Gerstein M, Snyder M (2009) RNA-seq: a revolutionary tool for transcriptomics.
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