Abstract
The placenta is the organ that dictates the reproductive outcome of mammalian pregnancy by supplying nutrients and oxygen to the developing fetus to sustain its normal growth. During early mammalian development, trophoblast cells are the earliest cell type to differentiate with multipotent capacity to generate the trophoblast components of the placenta. The isolation and use of mouse trophoblast stem cells (mTSCs) to model in vitro trophoblast differentiation, in combination with CRISPR/Cas9 genome editing technology, has provided tremendous insight into the molecular mechanisms governing early mouse placentation. By knocking out a specific gene of interest in mTSCs, researchers are shedding light onto the molecular pathways involved in normal placental development and pregnancy disorders associated with abnormal placentation. In this chapter, we provide a detailed protocol for the genetic modification of mTSCs by using CRISPR/Cas9 genome editing system.
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References
Brosens I, Pijnenborg R, Vercruysse L, Romero R (2011) The “Great Obstetrical Syndromes” are associated with disorders of deep placentation. Am J Obstet Gynecol 204(3):193–201. https://doi.org/10.1016/j.ajog.2010.08.009
Pijnenborg R, Vercruysse L, Brosens I (2011) Deep placentation. Best Pract Res Clin Obstet Gynaecol 25(3):273–285. https://doi.org/10.1016/j.bpobgyn.2010.10.009
Latos PA, Hemberger M (2016) From the stem of the placental tree: trophoblast stem cells and their progeny. Development 143(20):3650–3660. https://doi.org/10.1242/dev.133462
Tanaka S, Kunath T, Hadjantonakis AK, Nagy A, Rossant J (1998) Promotion of trophoblast stem cell proliferation by FGF4. Science 282(5396):2072–2075. https://doi.org/10.1126/science.282.5396.2072
Perez-Garcia V, Fineberg E, Wilson R, Murray A, Mazzeo CI, Tudor C et al (2018) Placentation defects are highly prevalent in embryonic lethal mouse mutants. Nature 555(7697):463–468. https://doi.org/10.1038/nature26002
Giuliano CJ, Lin A, Girish V, Sheltzer JM (2019) Generating single cell-derived knockout clones in mammalian cells with CRISPR/Cas9. Curr Protoc Mol Biol 128(1):e100. https://doi.org/10.1002/cpmb.100
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308. https://doi.org/10.1038/nprot.2013.143
Chrysanthou S, Senner CE, Woods L, Fineberg E, Okkenhaug H, Burge S et al (2018) A critical role of TET1/2 proteins in cell-cycle progression of trophoblast stem cells. Stem Cell Rep 10(4):1355–1368. https://doi.org/10.1016/j.stemcr.2018.02.014
Murray A, Sienerth AR, Hemberger M (2016) Plet1 is an epigenetically regulated cell surface protein that provides essential cues to direct trophoblast stem cell differentiation. Sci Rep 6:25112. https://doi.org/10.1038/srep25112
Perez-Garcia V, Lea G, Lopez-Jimenez P, Okkenhaug H, Burton GJ, Moffett A et al (2021) BAP1/ASXL complex modulation regulates epithelial-mesenchymal transition during trophoblast differentiation and invasion. Elife 10. https://doi.org/10.7554/eLife.63254
Ballasy NN, Bering EA, Kokorudz C, Radford BN, Zhao X, Dean W et al (2022) Padi2/3 deficiency alters the epigenomic landscape and causes premature differentiation of mouse trophoblast stem cells. Cells 11(16). https://doi.org/10.3390/cells11162466
Todd CD, Deniz Ö, Taylor D, Branco MR (2019) Functional evaluation of transposable elements as enhancers in mouse embryonic and trophoblast stem cells. Elife 8. https://doi.org/10.7554/eLife.44344
Branco MR, King M, Perez-Garcia V, Bogutz AB, Caley M, Fineberg E et al (2016) Maternal DNA methylation regulates early trophoblast development. Dev Cell 36(2):152–163. https://doi.org/10.1016/j.devcel.2015.12.027
Acknowledgments
PDB has a CIPF Predoctoral Training Fellowship supported by Janssen Pharmaceutics. Research in the VPG laboratory was supported by the Ministerio de Ciencia e Innovación, Agencia Estatal de Investigación (RYC-2019-026956, PID2020-114459RA-I00/AEI/10.13039/501100011033) and Fundación Ramón Areces. Part of the equipment employed in this work was funded by Generalitat Valenciana and cofinanced with ERDF funds (OP ERDF of Comunitat Valenciana 2014–2020). MH is supported by a Tier I Canada Research Chair in Developmental Genetics and Epigenetics. Figures were created with Biorender.com.
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Doria-Borrell, P., Moya-Navamuel, M., Hemberger, M., Pérez-García, V. (2024). Generation of Knockout Mouse Trophoblast Stem Cells by CRISPR/Cas9. In: Zaga-Clavellina, V. (eds) Maternal Placental Interface. Methods in Molecular Biology, vol 2781. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3746-3_8
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DOI: https://doi.org/10.1007/978-1-0716-3746-3_8
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