Abstract
Many applications in human pluripotent stem cell (PSC) research require the genetic modification of PSCs to express a transgene in a stable and dependable manner. Random transgene integration commonly results in unpredictable and heterogeneous expression. We describe a protocol for the derivation of clonal populations of human embryonic stem cells or induced pluripotent stem cells (iPSCs) expressing a transgene from a single copy of an integrated lentiviral vector that is mapped to the genome. Using optimized transduction conditions, followed by single-cell subcloning and a round of antibiotic selection, we find that approximately half of the colonies retrieved contain a single vector copy. After expansion, the majority of these are confirmed to be clonal. The vector/genomic DNA junction is sequenced and the unique integration site is mapped to the genome. This protocol enables the efficient derivation of genetically modified PSCs containing an integrated transgene at a known genomic site in ∼7 weeks.
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Acknowledgements
This work was supported by the Starr Foundation (Tri-Institutional Stem Cell Initiative, Tri-SCI-018); by New York State Stem Cell Science, NYSTEM (N08T-060); and by the National Heart, Blood and Lung Institute, NHLBI grant HL053750. We thank members of the Sadelain, Riviere, Studer and Tomishima laboratories (Memorial Sloan-Kettering Cancer Center, New York) for helpful discussions and technical assistance.
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E.P.P. developed the protocol and wrote the paper. M.S. supervised the study and edited the paper.
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Papapetrou, E., Sadelain, M. Derivation of genetically modified human pluripotent stem cells with integrated transgenes at unique mapped genomic sites. Nat Protoc 6, 1274–1289 (2011). https://doi.org/10.1038/nprot.2011.362
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DOI: https://doi.org/10.1038/nprot.2011.362
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