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Derivation of genetically modified human pluripotent stem cells with integrated transgenes at unique mapped genomic sites

Nature Protocols volume 6, pages 1274–1289 (2011)Cite this article

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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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Figure 1: Transduction with lentiviral vector to generate single-copy iPSC clones.
Figure 2: Transgene (eGFP) expression in iPSCs transduced with the TNS9.
Figure 3: Assessment of gene transfer after transduction.
Figure 4: Human iPSCs grown as colonies or single cells.
Figure 5: qPCR standards used for absolute quantification.
Figure 6: Analysis of clonality by Southern blotting.
Figure 7: Mapping the integration site by iPCR.
Figure 8: Example of sequencing read of an iPCR product.
Figure 9: Digested genomic DNA.

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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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  1. Center for Cell Engineering, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Eirini P Papapetrou & Michel Sadelain

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  1. Eirini P Papapetrou
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Contributions

E.P.P. developed the protocol and wrote the paper. M.S. supervised the study and edited the paper.

Corresponding author

Correspondence to Eirini P Papapetrou.

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The authors declare no competing financial interests.

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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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