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.
Keywords: Embryonic stem cells; Gene silencing; Lentiviral shRNA delivery; shRNA; shRNA screening.
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