A Simple, Rapid, and Cost-Effective Method for Loss-of-Function Assays in Pluripotent Cells.

Autor:	Covelo-Molares H; Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela (USC), Santiago de Compostela, Spain., Souto Y; Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela (USC), Santiago de Compostela, Spain., Fidalgo M; Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela (USC), Santiago de Compostela, Spain., Guallar D; Center for Research in Molecular Medicine and Chronic Diseases (CiMUS), Universidade de Santiago de Compostela (USC), Santiago de Compostela, Spain. diana.guallar@usc.es.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2022; Vol. 2520, pp. 199-213.
DOI:	10.1007/7651_2021_434
Abstrakt:	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. (© 2021. Springer Science+Business Media, LLC.)
Databáze:	MEDLINE
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