ArticleMethods in molecular biology (Clifton, N.J.)2027
Lentiviral Transduction of Embryonic Stem Cells.
Article in Methods in molecular biology (Clifton, N.J.), 2027. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Lentiviral vectors provide an efficient and reliable method for stable gene knockdown in embryonic stem cells (ESCs) through RNA interference. Here, we describe a detailed protocol for lentiviral transduction of mouse ESCs using lentiviral shRNA expression vectors. The protocol encompasses lentiviral particle production in HEK-293T packaging cells, determination of viral titer, transduction of ESCs cultured under feeder-free conditions, and selection of stably transduced cells. Additionally, we describe methods for evaluating transduction efficiency using fluorescence microscopy and flow cytometry, as well as for assessing gene knockdown efficacy by quantitative real-time PCR (Q-RT-PCR). This protocol is suitable for functional genomic studies in pluripotent stem cells and can be adapted for other difficult-to-transfect cell types.
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