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Curr Protoc Stem Cell Biol. 2012 May;Chapter 2:Unit 5C.2. doi: 10.1002/9780470151808.sc05c02s21.

High-efficiency transfection and siRNA-mediated gene knockdown in human pluripotent stem cells.

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  • 1Yale Stem Cell Center, School of Medicine, Yale University, New Haven, Connecticut, USA.

Abstract

This unit describes a protocol on how to achieve high transfection efficiency on human embryonic stem cells and induced pluripotent stem cells using the common transfection reagent Lipofectamine 2000 as a carrier instead of involving a virus, and/or expensive equipment and reagents. Applying this technique for siRNA-mediated gene targeting to knockdown genes in the pluripotent stem cells, the expression of pluripotent genes, such as OCT4 and LIN28, was downregulated by more than 90% in multiple pluripotent cell lines. Beyond reaching high transfection efficiency on pluripotent cells, this protocol should also have application to primary cells that are traditionally difficult to transfect.

PMID:
22605647
[PubMed - indexed for MEDLINE]
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