Abstract
Embryonic stem cells (ESCs) are derived from the inner cell mass of the preimplantation blastocyst and can be maintained indefinitely in vitro without losing their properties. Given their self-renewal and pluripotency, ESCs not only represent a key tool to study early embryonic development in a dish, but also an unlimited source of material for tissue replacement in regenerative medicine. Loss-of-function assays using RNA interference are a powerful tool to understand the roles of specific genes and are facilitated by lentiviral-mediated delivery of vector-encoded shRNAs which allows long-term silencing of single or multiple genes. Here, we describe the steps for rapid and cost-effective production and testing of lentiviral particles with vector-encoded shRNAs for gene silencing in ESCs. This protocol can be easily adapted for loss-of-function assays in other pluripotent cells or culture conditions of interest.
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Acknowledgments
This research was funded by grants from the Spanish Agencia Estatal de Investigación, cofunded by the FEDER Program of the EU (RTI2018-096708-J-I00/AEI/FEDER,UE to D.G.); and the 2020 Leonardo Grant for Researchers and Cultural Creators BBVA Foundation (IN[20]_BBM_BAS_0292 to D.G.). The research from the M.F. laboratory was supported by the Spanish Agencia Estatal de Investigación (PID2019-105739GB-I00/AEI and BFU2016-80899-P/AEI/FEDER,UE), the Xunta de Galicia-Consellería de Cultura, Educación e Ordenación Universitaria (ED431F 2016/016), and the Fundación Ramón Areces (2016-PO025 to M.F.). D.G. (RYC2019-027305-I) and M.F. (RYC-2014-16779) are recipients of a Ramón y Cajal Award from the MICI and the MINECO of Spain, respectively. Y.S. is a recipient of a predoctoral fellowship from Xunta de Galicia (ED481A-2017/166).
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Covelo-Molares, H., Souto, Y., Fidalgo, M., Guallar, D. (2021). A Simple, Rapid, and Cost-Effective Method for Loss-of-Function Assays in Pluripotent Cells. In: Turksen, K. (eds) Embryonic Stem Cell Protocols . Methods in Molecular Biology, vol 2520. Humana, New York, NY. https://doi.org/10.1007/7651_2021_434
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DOI: https://doi.org/10.1007/7651_2021_434
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