Identification of Potential Molecular Determinants of Murine Embryonic Stem Cell Differentiation by a Transposon-Based Approach
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Embryonic stem cells (ESCs) are self-renewing pluripotent cells, capable of differentiating into all somatic cell types. The molecular control of self-renewal is relatively well-characterized, whereas how ESCs exit pluripotent state to differentiate is poorly understood. Here we identify two genes are required for differentiation and dozens of intergenic regions that potentially regulate ESC differentiation. We used PiggyBac (PB) transposon-based approach to randomly mutate the genome of ESCs, and generated hundreds of clones that resisted differentiation signals. Each clone was sequenced to determine genomic regions mutated by PB insertion. Intriguingly, many mutations were localized among intergenic regions and we identified two genes are required for differentiation. This study should facilitate further exploration of novel molecular determinants of embryonic stem cell differentiation.
KeywordsEmbryonic stem cell Pluripotency Cell differentiation Stromal interaction molecule 1 (STIM1) Mitogen-activated protein kinase (MAPK) PiggyBac transposon
We thank Professor Pentao Liu (The Wellcome Trust Sanger Institute, United Kingdom) for kindly providing PiggyBac plasmid, Mrs. Ling Lin and the Science & Technology Department of West China Second University Hospital at Sichuan University for administrative assistance.
TQ and YF conceived the experiments. YW, TL, PD, and QD conducted experiments and participated in writing the manuscript. All authors reviewed the manuscript.
This work was supported by National Natural Science Foundation of China (81671115), and Science and Technology Department of Sichuan Province (2016JQ0029). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Compliance with Ethical Standards
Conflict of interest
The authors declare no competing financial interests.
- 9.Wang, W., Lin, C., Lu, D., Ning, Z., Cox, T., Melvin, D., Wang, X., Bradley, A., & Liu, P. (2008). Chromosomal transposition of PiggyBac in mouse embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America, 105, 9290–9295.CrossRefPubMedPubMedCentralGoogle Scholar
- 11.Yeom, Y. I., Fuhrmann, G., Ovitt, C. E., Brehm, A., Ohbo, K., Gross, M., Hubner, K., & Scholer, H. R. (1996). Germline regulatory element of Oct-4 specific for the totipotent cycle of embryonal cells. Development (Cambridge, England), 122, 881–894.Google Scholar
- 13.Uren, A. G., Mikkers, H., Kool, J., van der Weyden, L., Lund, A. H., Wilson, C. H., Rance, R., Jonkers, J., van Lohuizen, M., Berns, A., & Adams, D. J. (2009). A high-throughput splinkerette-PCR method for the isolation and sequencing of retroviral insertion sites. Nature Protocols, 4, 789–798.CrossRefPubMedPubMedCentralGoogle Scholar
- 15.Barruet, E., Hadadeh, O., Peiretti, F., Renault, V. M., Hadjal, Y., Bernot, D., Tournaire, R., Negre, D., Juhan-Vague, I., Alessi, M. C., & Binetruy, B. (2011). p38 mitogen activated protein kinase controls two successive-steps during the early mesodermal commitment of embryonic stem cells. Stem Cells and Development, 20, 1233–1246.CrossRefPubMedGoogle Scholar
- 28.Lei, T., Zhang, P., Zhang, X., Xiao, X., Zhang, J., Qiu, T., Dai, Q., Zhang, Y., Min, L., Li, Q., Yin, R., Ding, P., Li, N., Qu, Y., Mu, D., Qin, J., Zhu, X., Xiao, Z. X., & Li, Q. (2018). Cyclin K regulates prereplicative complex assembly to promote mammalian cell proliferation. Nature Communications, 9, 1876.CrossRefPubMedPubMedCentralGoogle Scholar