title: High-efficiency transfection and siRNA-mediated gene knockdown in human pluripotent stem cells.
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.
authors.affiliation: Yale Stem Cell Center, School of Medicine, Yale University, New Haven, Connecticut, USA.
authors.forename: Yinghong
authors.initials: Y
authors.lastname: Ma
authors.forename: Haifan
authors.initials: H
authors.lastname: Lin
authors.forename: Caihong
authors.initials: C
authors.lastname: Qiu
mesh_terms: D002478:Cells, Cultured; D003001:Cloning, Molecular; D003094:Collagen / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D004338:Drug Combinations; D061252:Feeder Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D055785:Gene Knockdown Techniques / Q000379:methods*; D006801:Humans; D007797:Laminin / Q000494:pharmacology; D010957:Plasmids / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011509:Proteoglycans / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
chemical_list: D016895:Culture Media, Serum-Free; D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; D034741:RNA, Small Interfering; C059485:matrigel; D003094:Collagen