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

  

Authors:	Papapetrou, E. P.; Sadelain, M.
Article Title:	Derivation of genetically modified human pluripotent stem cells with integrated transgenes at unique mapped genomic sites
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. © 2011 Nature America, Inc. All rights reserved.
Keywords:	signal transduction; controlled study; antibiotic agent; human cell; nonhuman; gene expression; embryo; embryonic stem cell; cell line; cell differentiation; genetic vectors; transduction, genetic; gene mapping; cryopreservation; dna modification; molecular sequence data; genetic engineering; lentivirus; lentivirus vector; transgene; cell culture techniques; base sequence; embryonic stem cells; dna sequence; pluripotent stem cell; gene dosage; cell clone; genomic dna; transgenes; genome, human; induced pluripotent stem cells; chromosome mapping; stem cell expansion; cell cloning; process optimization; dna integration; virus cultivation
Journal Title:	Nature Protocols
Volume:	6
Issue:	9
ISSN:	1754-2189
Publisher:	Nature Publishing Group
Date Published:	2011-08-04
Start Page:	1274
End Page:	1289
Language:	English
DOI:	10.1038/nprot.2011.362
PROVIDER:	scopus
PUBMED:	21886096
DOI/URL:	http://www.scopus.com/inward/record.url?eid=2-s2.0-80052398745&partnerID=40&md5=c73f6f1dd3f7ad6684d4732cabaee545
Notes:	--- - "Export Date: 3 October 2011" - "Source: Scopus"

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